BASB006 polypeptides from Neisseria meningitidis and immunogenic compositions thereof

ABSTRACT

Provided are BASB006 polypeptides from  Neisseria meningitidis.  Also provided are fusion proteins containing BASB006 polypeptides. The invention also relates to immunogenic compositions containing BASB006 polypeptides.

This application claims the priority of GB Application No. 9808866.9,filed Apr. 24, 1998.

FIELD OF THE INVENTION

This invention relates to polynucleotides, (herein referred to as“BASB006 polynucleotide(s)”), polypeptides encoded by them (referred toherein as “BASB006” or “BASB006 polypeptide(s)”), recombinant materialsand methods for their production. In another aspect, the inventionrelates to methods for using such polypeptides and polynucleotides,including vaccines against bacterial infections. In a further aspect,the invention relates to diagnostic assays for detecting infection ofcertain pathogens.

BACKGROUND OF THE INVENTION

Neisseria meningitidis (meningococcus) is a Gram negative bacteriumfrequently isolated from the human upper respiratory tract. Itoccasionally causes invasive bacterial diseases such as bacteremia andmeningitis. The incidence of meningococcal disease shows geographicalseasonal and annual differences (Schwartz, B., Moore, P. S., Broome, C.V.; Clin. Microbiol. Rev. 2 (Supplement), S18-S24, 1989). Most diseasein temperate countries is due to strains of serogroup B and varies inincidence from 1-10/100,000/year total population sometimes reachinghigher values (Kaczmarski, E. B. (1997), Commun. Dis. Rep. Rev. 7:R55-9, 1995; Scholten, R. J. P. M., Bijlmer, H. A., Poolman, J. T. etal. Clin. Infect. Dis. 16: 237-246, 1993; Cruz, C., Pavez, G., Aguilar,E., et al. Epidemiol. Infect. 105: 119-126, 1990).

Epidemics dominated by serogroup. A meningococci, mostly in centralAfrica, are encountered, sometimes reaching levels up to1000/100.000/year (Schwartz, B., Moore. P. S., Broome, C. V. Clin.Microbiol. Rev. 2 (Supplement), S18-S24, 1989). Nearly all cases as awhole of meningococcal disease are caused by serogroup A, B, C, W-135and Y meningococci and a tetravalent A, C, W-135, Y polysaccharidevaccine is available (Armand, J., Arminjon, F., Mynard, M. C., Lafaix,C., J. Biol. Stand. 10: 335-339, 1982).

The polysaccharide vaccines are currently being improved by way ofchemical conjugating them to carrier proteins (Lieberman, J. M., Chiu,S. S., Wong, V. K., et al. JAMA 275: 1499-1503, 1996).

A serogroup B vaccine is not available, since the B capsularpolysaccharide was found to be nonimmunogenic, most likely because itshares structural similarity to host components (Wyle. F. A.,Artenstein. M. S., Brandt, M. L. et al. J. Infect. Dis. 126: 514-522.1972; Finne. J. M., Leinonen, M., Mäkelä, P. M. Lancet ii.: 355-357,1983).

For many years efforts have been initiated and carried out to developmeningococcal outer membrane based vaccines (de Moraes, J. C., Perkins,B., Camargo, M. C. et al. Lancet 340: 1074-1078, 1992; Bjune, G., Hoiby,E. A. Gronnesby, J. K. et al. 338: 1093-1096, 1991). Such vaccines havedemonstrated efficcacies from 57%-85% in older children (>4 years) andadolescents.

Many bacterial outer membrane components are present in these vaccines,such as PorA, PorB, Rmp, Opc, Opa, FrpB and the contribution of thesecomponents to the observed protection still needs further definition.Other bacterial outer membrane components have been defined by usinganimal or human antibodies to be potentially relevant to the inductionof protective immunity, such as TbpB and NspA (Martin, D., Cadieux, N.,Hamel, J., Brodeux, B. R., J. Exp. Med. 185: 1173-1183, 1997; Lissolo,L., Maitre-Wilmotte, C., Dumas, p. et al., Inf. Immnun. 63: 884-890,1995). The mechanisms of protective immunity will involve antibodymediated bactericidal activity and opsonophagocytosis.

A bacteremia animal model has been used to combine all antibody mediatedmechanisms (Saukkonen, K., Leinonen, M., Abdillahi, H. Poolman, J. T.Vaccine 7: 325-328, 1989). It is generally accepted that the latecomplement component mediated bactericidal mechanism is crucial forimmunity against meningococcal disease (Ross. S. C., Rosenthal P. J.,Berberic, H. M., Densen, P. J. Infect. Dis. 155: 1266-1275. 1987).

The frequency of Neisseria meningitidis infections has risendramatically in the past few decades. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Neisseria meningitidis strains that are resistant tosome or all of the standard antibiotics. This phenomenon has created anunmet medical need and demand for new anti-microbial agents, vaccines,drug screening methods, and diagnostic tests for this organism.

SUMMARY OF THE INVENTION

The present invention relates to BASB006, in particular BASB006polypeptides and BASB006 polynucleotides, recombinant materials andmethods for their production. In another aspect, the invention relatesto methods for using such polypeptides and polynucleotides, includingprevention and treatment of microbial diseases, amongst others. In afurther aspect, the invention relates to diagnostic assays for detectingdiseases associated with microbial infections and conditions associatedwith such infections, such as assays for detecting expression oractivity of BASB006 polynucleotides or polypeptides.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

FIGS. 1A-1L show consecutive segments of sequence alignments for twoBASB006-encoding polynucleotides.

FIGS. 2A-2D show consecutive segments of sequence alignments for twoBASB006 polypeptides.

FIG. 3 shows a Coomassie stain SDS-PAGE gel analysis of purified BASB006protein.

FIG. 4 shows a Western-blot of partially purified recombinant BASB006protein probed with mice sera containing anti-BASB006 antibodies.

FIG. 5 shows a Western-blot of partially purified recombinant BASB006protein probed with human convalescent sera and mice sera.

DESCRIPTION OF THE INVENTION

The invention relates to BASB006 polypeptides and polynucleotides asdescribed in greater detail below. In particular, the invention relatesto polypeptides and polynucleotides of BASB006 of Neisseriameningitidis, which is related by amino acid sequence homology to H.influenzae Hap polypeptide. The invention relates especially to BASB006having the nucleotide and amino acid sequences set out in SEQ ID NO:13and SEQ ID NO:2,4 respectively. It is understood that sequences recitedin the Sequence Listing below as “DNA” represent an exemplification ofone embodiment of the invention. since those of ordinary skill willrecognize that such sequences can be usefully employed inpolynucleotides in general, including ribopolynucleotides.

Polypeptides

In one aspect of the invention there are provided polypeptides ofNeisseria meningitidis referred to herein as “BASB006” and “BASB006polypeptides” as well as biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

The present invention further provides for:

(a) an isolated polypeptide which comprises an amino acid sequence whichhas at least 85% identity, more preferably at least 90% identity, yetmore preferably at least 95% identity, most preferably at least 97-99%or exact identity, to that of SEQ ID NO:2, 4;

(b) a polypeptide encoded by an isolated polynucleotide comprising apolynucleotide sequence which has at least 85% identity, more preferablyat least 90% identity, yet more preferably at least 95% identity, evenmore preferably at least 97-99% or exact identity to SEQ ID NO:1, 3 overthe entire length of SEQ ID NO:1, 3 respectively; or

(c) a polypeptide encoded by an isolated polynucleotide comprising apolynucleotide sequence encoding a polypeptide which has at least 85%identity, more preferably at least 90% identity, yet more preferably atleast 95% identity, even more preferably at least 97-99% or exactidentity, to the amino acid sequence of SEQ ID NO:2. 4;

The BASB006 polypeptides provided in SEQ ID NO:2,4 are the BASB006polypeptides from Neisseria meningitidis strains American Type CultureCollection 13090 (herein “ATCC 13090”) and H44/76.

The invention also provides an immunogenic fragment of a BASB006polypeptide, that is, a contiguous portion of the BASB006 polypeptidewhich has the same or substantially the same immunogenic activity as thepolypeptide comprising the amino acid sequence of SEQ ID NO:2,4. That isto say, the fragment (if necessary when coupled to a carrier) is capableof raising an immune response which recognises the BASB006 polypeptideSuch an immunogenic fragment may include, for example, the BASB006polypeptide lacking an N-terminal leader sequence, and/or atransmembrane domain and/or a C-terminal anchor domain. In a preferredaspect the immunogenic fragment of BASB006 according to the inventioncomprises substantially all of the extracellular domain of a polypeptidewhich has at least 85% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, most preferably at least97-99% identity, to that of SEQ ID NO:2,4 over the entire length of SEQID NO:2

A fragment is a polypeptide having an amino acid sequence that isentirely the same as part but not all of any amino acid sequence of anypolypeptide of the invention. As with BASB006 polypeptides, fragmentsmay be “free-standing” or comprised within a larger polypeptide of whichthey form a part or region, most preferably- as a single continuousregion in a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of an amino acid sequence of SEQ ID NO:2,4 or of variantsthereof, such as a continuous series of residues that includes an amino-and/or carboxyl-terminal amino acid sequence.

Degradation forms of the polypeptides of the invention produced by or ina host cell, are also preferred. Further preferred are fragmentscharacterized by structural or functional attributes such as fragmentsthat comprise alpha-helix and alpha-helix forming regions, beta-sheetand beta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions.

Further preferred fragments include an isolated polypeptide comprisingan amino acid sequence having at least 15, 20, 30, 40, 50 or 100contiguous amino acids from the amino acid sequence of SEQ ID NO:2,4, oran isolated polypeptide comprising an amino acid sequence having atleast 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated ordeleted from the amino acid sequence of SEQ ID NO:2,4.

Fragments of the polypeptides of the invention may be employed forproducing the corresponding full-length polypeptide by peptidesynthesis; therefore, these fragments may be employed as intermediatesfor producing the full-length polypeptides of the invention.

Particularly preferred are variants in which several, 5-10, 1-5, 1-3,1-2 or 1 amino acids are substituted, deleted, or added in anycombination.

The polypeptides, or immunogenic fragments, of the invention may be inthe form of the “mature” protein or may be a part of a larger proteinsuch as a precursor or a fusion protein. It is often advantageous toinclude an additional amino acid sequence which contains secretory orleader sequences, pro-sequences, sequences which aid in purificationsuch as multiple histidine residues, or an additional sequence forstability during recombinant production. Furthermore, addition ofexogenous polypeptide or lipid tail or polynucleotide sequences toincrease the immunogenic potential of the final molecule is alsoconsidered.

In one aspect, the invention relates to genetically engineered solublefusion proteins comprising a polypeptide of the present invention, or afragment thereof, and various portions of the constant regions of heavyor light chains of immunoglobulins of various subclasses (IgG, Ig, IgA,IgE). Preferred as an immunoglobulin is the constant part of the heavychain of human IgG, particularly IgG 1, where fusion takes place at thehinge region. In a particular embodiment, the Fc part can be removedsimply by incorporation of a cleavage sequence which can be cleaved withblood clotting factor Xa.

Furthermore, this invention relates to processes for the preparation ofthese fusion proteins by genetic engineering, and to the use thereof fordrug screening, diagnosis and therapy. A further aspect of the inventionalso relates to polynucleotides encoding such fusion proteins. Examplesof fusion protein technology can be found in International PatentApplication Nos. WO94/29458 and WO94/22914.

The proteins may be chemically conjugated, or expressed as recombinantfusion proteins allowing increased levels to be produced in anexpression system as compared to non-fused protein. The fusion partnermay assist in providing T helper epitopes (immunological fusionpartner), preferably T helper epitopes recognised by humans, or assistin expressing the protein (expression enhancer) at higher yields thanthe native recombinant protein. Preferably the fusion partner will beboth an immunological fusion partner and expression enhancing partner.

Fusion partners include protein D from Haemophilus influenzae and thenon-structural protein from influenzae virus, NS1 (hemagglutinin).Another fusion partner is the protein known as LytA. Preferably the Cterminal portion of the molecule is used. LytA is derived fromStreptococcus pneumoniae which synthesize an N-acetyl-L-alanine amidase,amidase LytA, (coded by the lytA gene {Gene, 43 (1986) page 265-272}) anautolysin that specifically degrades certain bonds in the peptidoglycanbackbone. The C-terminal domain of the LytA protein is responsible forthe affinity to the choline or to some choline analogues such as DEAE.This property has been exploited for the development of E. coli C-LytAexpressing plasmids useful for expression of fusion proteins.Purification of hybrid proteins containing the C-LytA fragment at itsamino terminus has been described {Biotechnology: 10, (1992) page795-798}. It is possible to use the repeat portion of the LytA moleculefound in the C terminal end starting at residue 178, for exampleresidues 188-305.

The present invention also includes variants of the aforementionedpolypeptides, that is polypeptides that vary from the referents byconservative amino acid substitutions, whereby a residue is substitutedby another with like characteristics. Typical such substitutions areamong Ala, Val, Leu and Ile; among Ser and Thr; among the acidicresidues Asp and Glu; among Asn and Gln; and among the basic residuesLys and Arg; or aromatic residues Phe and Tyr.

Polypeptides of the present invention can be prepared in any suitablemanner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

It is most preferred that a polypeptide of the invention is derived fromNeisseria meningitidis, however, it may preferably be obtained fromother organisms of the same taxonomic genus. A polypeptide of theinvention may also be obtained, for example, from organisms of the sametaxonomic family or order.

Polynucleotides

It is an object of the invention to provide polynucleotides that encodeBASB006 polypeptides, particularly polynucleotides that encode thepolypeptide herein designated BASB006.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding BASB006 polypeptidescomprising a sequence set out in SEQ ID NO:1,3 which includes a fulllength gene, or a variant thereof.

The BASB006 polynucleotides provided in SEQ ID NO:1,3 are the BASB006polynucleotides from Neisseria meningitidis strains ATCC 13090 andH44/76.

As a further aspect of the invention there are provided isolated nucleicacid molecules encoding and/or expressing BASB006 polypeptides andpolynucleotides, particularly Neisseria meningitidis BASB006polypeptides and polynucleotides, including, for example, unprocessedRNAs, ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs. Furtherembodiments of the invention include biologically, diagnostically,prophylactically, clinically or therapeutically useful polynucleotidesand polypeptides, and variants thereof, and compositions comprising thesame.

Another aspect of the invention relates to isolated polynucleotides,including at least one full length gene, that encodes a BASB006polypeptide having a deduced amino acid sequence of SEQ ID NO:2,4 andpolynucleotides closely related thereto and variants thereof.

In another particularly preferred embodiment of the invention there is aBASB006 polypeptide from Neisseria meningitidis comprising or consistingof an amino acid sequence of SEQ ID NO:2,4 or a variant thereof.

Using the information provided herein, such as a polynucleotide sequenceset out in SEQ ID NO:1, 3 a polynucleotide of the invention encodingBASB006 polypeptide may be obtained using standard cloning and screeningmethods, such as those for cloning and sequencing chromosomal DNAfragments from bacteria using Neisseria meningitidis cells as startingmaterial, followed by obtaining a full length clone. For example, toobtain a polynucleotide sequence of the invention, such as apolynucleotide sequence given in SEQ ID NO:1,3, typically a library ofclones of chromosomal DNA of Neisseria meningitidis in E.coli or someother suitable host is probed with a radiolabeled oligonucleotide,preferably a 17-mer or longer, derived from a partial sequence. Clonescarrying DNA identical to that of the probe can then be distinguishedusing stringent hybridization conditions. By sequencing the individualclones thus identified by hybridization with sequencing primers designedfrom the original polypeptide or polynucleotide sequence it is thenpossible to extend the polynucleotide sequence in both directions todetermine a full length gene sequence. Conveniently, such sequencing isperformed for example, using denatured double stranded DNA prepared froma plasmid clone. Suitable techniques are described by Maniatis. T.,Fritsch, E. F. and Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989). (see in particular Screening By Hybridization 1.90and Sequencing Denatured Double-Stranded DNA Templates 13.70). Directgenomic DNA sequencing may also be performed to obtain a full lengthgene sequence. Illustrative of the invention, each polynucleotide setout in SEQ ID NO:1,3 was discovered in a DNA library derived fromNeisseria meningitidis.

Moreover, each DNA sequence set out in SEQ ID NO:1,3 contains an openreading frame encoding a protein having about the number of amino acidresidues set forth in SEQ ID NO:2, 4 with a deduced molecular weightthat can be calculated using amino acid residue molecular weight valueswell known to those skilled in the art.

The polynucleotide of SEQ ID NO:1, between the start codon at nucleotidenumber 1 and the stop codon which begins at nucleotide number 4363 ofSEQ ID NO:1, encodes the polypeptide of SEQ ID NO:2.

The polynucleotide of SEQ ID NO:3, between the start codon at nucleotidenumber 1 and the stop codon which begins at nucleotide number 4372 ofSEQ ID NO:3, encodes the polypeptide of SEQ ID NO:4.

In a further aspect, the present invention provides for an isolatedpolynucleotide comprising or consisting of:

(a) a polynucleotide sequence which has at least 85% identity, morepreferably at least 90% identity, yet more preferably at least 95%identity, even more preferably at least 97-99% or exact identity to SEQID NO:1,3 over the entire length of SEQ ID NO:1,3 respectively; or

(b) a polynucleotide sequence encoding a polypeptide which has at least85% identity, more preferably at least 90% identity, yet more preferablyat least 95% identity, even more preferably at least 97-99% or 100%exact, to the amino acid sequence of SEQ ID NO:2, 4 over the entirelength of SEQ ID NO:2, 4 respectively.

A polynucleotide encoding a polypeptide of the present invention,including homologs and orthologs from species other than Neisseriameningitidis, may be obtained by a process which comprises the steps ofscreening an appropriate library under stringent hybridizationconditions (for example, using a temperature in the range of 45-65° C.and an SDS concentration from 0.1-1%) with a labeled or detectable probeconsisting of or comprising the sequence of SEQ ID NO:1, 3 or a fragmentthereof; and isolating a full-length gene and/or genomic clonescontaining said polynucleotide sequence.

The invention provides a polynucleotide sequence identical over itsentire length to a coding sequence (open reading frame) in SEQ ID NO:1,3. Also provided by the invention is a coding sequence for a maturepolypeptide or a fragment thereof, by itself as well as a codingsequence for a mature polypeptide or a fragment in reading frame withanother coding sequence, such as a sequence encoding a leader orsecretory sequence, a pre-, or pro- or prepro-protein sequence. Thepolynucleotide of the invention may also contain at least one non-codingsequence, including for example, but not limited to at least onenon-coding 5′ and 3′ sequence, such as the transcribed butnon-translated sequences, termination signals (such as rho-dependent andrho-independent termination signals), ribosome binding sites, Kozaksequences, sequences that stabilize mRNA, introns, and polyadenylationsignals. The polynucleotide sequence may also comprise additional codingsequence encoding additional amino acids. For example, a marker sequencethat facilitates purification of the fused polypeptide can be encoded.In certain embodiments of the invention, the marker sequence is ahexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) anddescribed in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824(1989), or an HA peptide tag (Wilson et al., Cell 37: 767 (1984), bothof which may be useful in purifying polypeptide sequence fused to them.Polynucleotides of the invention also include, but are not limited to,polynucleotides comprising a structural gene and its naturallyassociated sequences that control gene expression.

The nucleotide sequence encoding BASB006 polypeptide of SEQ ID NO:2, 4may be identical to the polypeptide encoding sequence contained innucleotides 1 to 4362 of SEQ ID NO:1, or the polypeptide encodingsequence contained in nucleotides 1 to 4371 of SEQ ID NO:3,respectively. Alternatively it may be a sequence, which as a result ofthe redundancy (degeneracy) of the genetic code, also encodes thepolypeptide of SEQ ID NO:2, 4.

The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Neisseria meningitidis BASB006having an amino acid sequence set out in SEQ ID NO:2, 4. The term alsoencompasses polynucleotides that include a single continuous region ordiscontinuous regions encoding the polypeptide (for example,polynucleotides interrupted by integrated phage, an integrated insertionsequence, an integrated vector sequence, an integrated transposonsequence, or due to RNA editing or genomic DNA reorganization) togetherwith additional regions, that also may contain coding and/or non-codingsequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode variants of a polypeptide having a deducedamino acid sequence of SEQ ID NO:2, 4. Fragments of polynucleotides ofthe invention may be used, for example, to synthesize full-lengthpolynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodingBASB006 variants, that have the amino acid sequence of BASB006polypeptide of SEQ ID NO:2, 4 in which several, a few, 5 to 10, 1 to 5,1 to 3, 2, 1 or no amino acid residues are substituted. modified,deleted and/or added, in any combination. Especially preferred amongthese are silent substitutions, additions and deletions, that do notalter the properties and activities of BASB006 polypeptide.

Further preferred embodiments of the invention are polynucleotides thatare at least 85% identical over their entire length to a polynucleotideencoding BASB006 polypeptide having an amino acid sequence set out inSEQ ID NO:2, 4, and polynucleotides that are complementary to suchpolynucleotides. In this regard, polynucleotides at least 90% identicalover their entire length to the same are particularly preferred, andamong these particularly preferred polynucleotides, those with at least95% are especially preferred.

Furthermore, those with at least 97% are highly preferred among thosewith at least 95%, and among these those with at least 98% and at least99% are particularly highly preferred, with at least 99% being the morepreferred.

Preferred embodiments are polynucleotides encoding polypeptides thatretain substantially the same biological function or activity as themature polypeptide encoded by a DNA of SEQ ID NO:1,3.

In accordance with certain preferred embodiments of this invention thereare provided polynucleotides that hybridize, particularly understringent conditions, to BASB006 polynucleotide sequences, such as thosepolynucleotides in SEQ ID NO:1, 3.

The invention further relates to polynucleotides that hybridize to thepolynucleotide sequences provided herein. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the polynucleotides described herein. As herein used, theterms “stringent conditions” and “stringent hybridization conditions”mean hybridization occurring only if there is at least 95% andpreferably at least 97% identity between the sequences. A specificexample of stringent hybridization conditions is overnight incubation at42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml of denatured,sheared salmon sperm DNA, followed by washing the hybridization supportin 0.1×SSC at about 65° C. Hybridization and wash conditions are wellknown and exemplified in Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989),particularly Chapter 11 therein. Solution hybridization may also be usedwith the polynucleotide sequences provided by the invention.

The invention also provides a polynucleotide consisting of or comprisinga polynucleotide sequence obtained by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set forth inSEQ ID NO:1, 3 under stringent hybridization conditions with a probehaving the sequence of said polynucleotide sequence set forth in SEQ IDNO:1, 3 or a fragment thereof; and isolating said polynucleotidesequence. Fragments useful for obtaining such a polynucleotide include,for example, probes and primers fully described elsewhere herein.

As discussed elsewhere herein regarding polynucleotide assays of theinvention, for instance, the polynucleotides of the invention, may beused as a hybridization probe for RNA. cDNA and genomic DNA to isolatefull-length cDNAs and genomic clones encoding BASB006 and to isolatecDNA and genomic clones of other genes that have a high identity,particularly high sequence identity, to the BASB006 gene. Such probesgenerally will comprise at least 15 nucleotide residues or base pairs.Preferably, such probes will have at least 30 nucleotide residues orbase pairs and may have at least 50 nucleotide residues or base pairs.Particularly preferred probes will have at least 20 nucleotide residuesor base pairs and will have less than 30 nucleotide residues or basepairs.

A coding region of a BASB006 gene may be isolated by screening using aDNA sequence provided in SEQ ID NO:1, 3 to synthesize an oligonucleotideprobe. A labeled oligonucleotide having a sequence complementary to thatof a gene of the invention is then used to screen a library of cDNA,genomic DNA or mRNA to determine which members of the library the probehybridizes to.

There are several methods available and well known to those skilled inthe art to obtain full-length DNAs, or extend short DNAs, for examplethose based on the method of Rapid Amplification of cDNA ends (RACE)(see, for example, Frohman, et al., PNAS USA 85: 8998-9002, 1988).Recent modifications of the technique, exemplified by the Marathon™technology (Clontech Laboratories Inc.) for example, have significantlysimplified the search for longer cDNAs. In the Marathon™ technology,cDNAs have been prepared from mRNA extracted from a chosen tissue and an‘adaptor’ sequence ligated onto each end. Nucleic acid amplification(PCR) is then carried out to amplify the “missing” 5′ end of the DNAusing a combination of gene specific and adaptor specificoligonucleotide primers. The PCR reaction is then repeated using“nested” primers, that is, primers designed to anneal within theamplified product (typically an adaptor specific primer that annealsfurther 3′ in the adaptor sequence and a gene specific primer thatanneals further 5′ in the selected gene sequence). The products of thisreaction can then be analyzed by DNA sequencing and a full-length DNAconstructed either by joining the product directly to the existing DNAto give a complete sequence, or carrying out a separate full-length PCRusing the new sequence information for the design of the 5′ primer.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for diseases, particularly human diseases,as further discussed herein relating to polynucleotide assays.

The polynucleotides of the invention that are oligonucleotides derivedfrom a sequence of SEQ ID NOS: 1-4 may be used in the processes hereinas described, but preferably for PCR, to determine whether or not thepolynucleotides identified herein in whole or in part are transcribed inbacteria in infected tissue. It is recognized that such sequences willalso have utility in diagnosis of the stage of infection and type ofinfection the pathogen has attained.

The invention also provides polynucleotides that encode a polypeptidethat is the mature protein plus additional amino or carboxyl-terminalamino acids, or amino acids interior to the mature polypeptide (when themature form has more than one polypeptide chain, for instance). Suchsequences may play a role in processing of a protein from precursor to amature form, may allow protein transport, may lengthen or shortenprotein half-life or may facilitate manipulation of a protein for assayor production, among other things. As generally is the case in vivo, theadditional amino acids may be processed away from the mature protein bycellular enzymes.

For each and every polynucleotide of the invention there is provided apolynucleotide complementary to it. It is preferred that thesecomplementary polynucleotides are fully complementary to eachpolynucleotide with which they are complementary.

A precursor protein, having a mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In addition to the standard A, G, C, T/U representations fornucleotides, the term “N” may also be used in describing certainpolynucleotides of the invention. “N” means that any of the four DNA orRNA nucleotides may appear at such a designated position in the DNA orRNA sequence, except it is preferred that N is not a nucleic acid thatwhen taken in combination with adjacent nucleotide positions, when readin the correct reading frame, would have the effect of generating apremature termination codon in such reading frame.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

In accordance with an aspect of the invention, there is provided the useof a polynucleotide of the invention for therapeutic or prophylacticpurposes, in particular genetic immunization.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolffet cl., Hum Mol Genet (1992)1: 363, Manthorpe et al., Hum. Gene Ther. (1983) 4: 419), delivery ofDNA complexed with specific protein carriers (Wu et al., J Biol Chem.(1989) 264: 16985), coprecipitation of DNA with calcium phosphate(Benvenisty & Reshef, PNAS USA, (1986) 83: 9551), encapsulation of DNAin various forms of liposomes (Kaneda et al., Science (1989) 243: 375),particle bombardment (Tang et al., Nature (1992) 356:152, Eisenbraun etal., DNA Cell Biol (1993) 12: 791) and in vivo infection using clonedretroviral vectors (Seeger et al., PNAS USA (1984) 81: 5849).

Vectors, Host Cells, Expression Systems

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

Recombinant polypeptides of the present invention may be prepared byprocesses well known in those skilled in the art from geneticallyengineered host cells comprising expression systems. Accordingly, in afurther aspect, the present invention relates to expression systems thatcomprise a polynucleotide or polynucleotides of the present invention,to host cells which are genetically engineered with such expressionsystems, and to the production of polypeptides of the invention byrecombinant techniques.

For recombinant production of the polypeptides of the invention, hostcells can be genetically engineered to incorporate expression systems orportions thereof or polynucleotides of the invention. Introduction of apolynucleotide into the host cell can be effected by methods describedin many standard laboratory manuals, such as Davis. et al., BASICMETHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook. et al., MOLECULARCLONING: A LABORATORY MANUAL. 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphatetransfection, DEAE-dextran mediated transfection, transvection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction and infection.

Representative examples of appropriate hosts include bacterial cells,such as cells of streptococci, staphylococci, enterococci, E. coli,streptomyces, cyanobacteria, Bacillus subtilis, Moraxella catarrhalis,Haemophilus influenzae and Neisseria meningitidis; fungal cells, such ascells of a yeast, Kluyveromyces, Saccharomyces, a basidiomycete, Candidaalbicans and Aspergillus; insect cells such as cells of Drosophila S2and Spodoptera Sf9; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK,293, CV-1 and Bowes melanoma cells; and plant cells, such as cells of agymnosperm or angiosperm.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal-, episomal- and virus-derived vectors, for example, vectorsderived from bacterial plasmids, from bacteriophage, from transposons,from yeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses, picomaviruses, retroviruses, and alphaviruses and vectorsderived from combinations thereof, such as those derived from plasmidand bacteriophage genetic elements, such as cosmids and phagemids. Theexpression system constructs may contain control regions that regulateas well as engender expression. Generally, any system or vector suitableto maintain, propagate or express polynucleotides and/or to express apolypeptide in a host may be used for expression in this regard. Theappropriate DNA sequence may be inserted into the expression system byany of a variety of well-known and routine techniques, such as, forexample, those set forth in Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, (supra).

In recombinant expression systems in eukaryotes, for secretion of atranslated protein into the lumen of the endoplasmic reticulum, into theperiplasmic space or into the extracellular environment, appropriatesecretion signals may be incorporated into the expressed polypeptide.These signals may be endogenous to the polypeptide or they may beheterologous signals.

Polypeptides of the present invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, ion metalaffinity chromatography (IMAC) is employed for purification. Well knowntechniques for refolding proteins may be employed to regenerate activeconformation when the polypeptide is denatured during intracellularsynthesis, isolation and or purification.

The expression system may also be a recombinant live microorganism, suchas a virus or bacterium. The gene of interest can be inserted into thegenome of a live recombinant virus or bacterium. Inoculation and in vivoinfection with this live vector will lead to in vivo expression of theantigen and induction of immune responses. Viruses and bacteria used forthis purpose are for instance: poxviruses (e.g; vaccinia. fowlpox,canarypox), alphaviruses (Sindbis virus, Semliki Forest Virus.Venezuelian Equine Encephalitis Virus), adenoviruses, adeno-associatedvirus, picornaviruses (poliovirus, rhinovirus), herpesviruses (varicellazoster virus, etc), Listeria, Salmonella , Shigella, Neisseria, BCG.These viruses and bacteria can be virulent, or attenuated in variousways in order to obtain live vaccines. Such live vaccines also form partof the invention.

Diagnostic, Prognostic, Serotyping and Mutation Assays

This invention is also related to the use of BASB006 polynucleotides andpolypeptides of the invention for use as diagnostic reagents. Detectionof BASB006 polynucleotides and/or polypeptides in a eukaryote,particularly a mammal, and especially a human, will provide a diagnosticmethod for diagnosis of disease, staging of disease or response of aninfectious organism to drugs. Eukaryotes, particularly mammals, andespecially humans, particularly those infected or suspected to beinfected with an organism comprising the BASB006 gene or protein, may bedetected at the nucleic acid or amino acid level by a variety of wellknown techniques as well as by methods provided herein.

Polypeptides and polynucleotides for prognosis, diagnosis or otheranalysis may be obtained from a putatively infected and/or infectedindividual's bodily materials. Polynucleotides from any of thesesources, particularly DNA or RNA, may be used directly for detection ormay be amplified enzymatically by using PCR or any other amplificationtechnique prior to analysis. RNA, particularly mRNA, cDNA and genomicDNA may also be used in the same ways. Using amplification,characterization of the species and strain of infectious or residentorganism present in an individual, may be made by an analysis of thegenotype of a selected polynucleotide of the organism. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to a genotype of a reference sequence selected from arelated organism, preferably a different species of the same genus or adifferent strain of the same species. Point mutations can be identifiedby hybridizing amplified DNA to labeled BASB006 polynucleotidesequences. Perfectly or significantly matched sequences can bedistinguished from imperfectly or more significantly mismatched duplexesby DNase or RNase digestion, for DNA or RNA respectively, or bydetecting differences in melting temperatures or renaturation kinetics.Polynucleotide sequence differences may also be detected by alterationsin the electrophoretic mobility of polynucleotide fragments in gels ascompared to a reference sequence. This may be carried out with orwithout denaturing agents. Polynucleotide differences may also bedetected by direct DNA or RNA sequencing. See, for example, Myers etal., Science, 230: 1242 (1985). Sequence changes at specific locationsalso may be revealed by nuclease protection assays, such as RNase, V1and S1 protection assay or a chemical cleavage method. See, for example,Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985).

In another embodiment, an array of oligonucleotides probes comprisingBASB006 nucleotide sequence or fragments thereof can be constructed toconduct efficient screening of, for example, genetic mutations,serotype, taxonomic classification or identification. Array technologymethods are well known and have general applicability and can be used toaddress a variety of questions in molecular genetics including geneexpression, genetic linkage, and genetic variability (see, for example,Chee et al., Science, 274: 610 (1996)).

Thus in another aspect, the present invention relates to a diagnostickit which comprises:

(a) a polynucleotide of the present invention, preferably the nucleotidesequence of SEQ ID NO:1, 3, or a fragment thereof;

(b) a nucleotide sequence complementary to that of (a);

(c) a polypeptide of the present invention, preferably the polypeptideof SEQ ID NO:2, 4 or a fragment thereof; or

(d) an antibody to a polypeptide of the present invention, preferably tothe polypeptide of SEQ ID NO:2, 4.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component. Such a kit will be of use indiagnosing a disease or susceptibility to a disease, among others.

This invention also relates to the use of polynucleotides of the presentinvention as diagnostic reagents. Detection of a mutated form of apolynucleotide of the invention, preferable, SEQ ID NO:1, 3 which isassociated with a disease or pathogenicity will provide a diagnostictool that can add to, or define, a diagnosis of a disease, a prognosisof a course of disease, a determination of a stage of disease, or asusceptibility to a disease, which results from under-expression,over-expression or altered expression of the polynucleotide. Organisms,particularly infectious organisms, carrying mutations in suchpolynucleotide may be detected at the polynucleotide level by a varietyof techniques, such as those described elsewhere herein.

Cells from an organism carrying mutations or polymorphisms (allelicvariations) in a polynucleotide and/or polypeptide of the invention mayalso be detected at the polynucleotide or polypeptide level by a varietyof techniques, to allow for serotyping, for example. For example, RT-PCRcan be used to detect mutations in the RNA. It is particularly preferredto use RT-PCR in conjunction with automated detection systems, such as,for example, GeneScan. RNA, cDNA or genomic DNA may also be used for thesame purpose, PCR. As an example, PCR primers complementary to apolynucleotide encoding BASB006 polypeptide can be used to identify andanalyze mutations.

The invention further provides primers with 1, 2, 3 or 4 nucleotidesremoved from the 5′ and/or the 3′ end. These primers may be used for,among other things, amplifying BASB006 DNA and/or RNA isolated from asample derived from an individual, such as a bodily material. Theprimers may be used to amplify a polynucleotide isolated from aninfected individual, such that the polynucleotide may then be subject tovarious techniques for elucidation of the polynucleotide sequence. Inthis way, mutations in the polynucleotide sequence may be detected andused to diagnose and/or prognose the infection or its stage or course,or to serotype and/or classify the infectious agent.

The invention further provides a process for diagnosing disease,preferably bacterial infections, more preferably infections caused byNeisseria meningitidis, comprising determining from a sample derivedfrom an individual, such as a bodily material, an increased level ofexpression of polynucleotide having a sequence of SEQ ID NO:1, 3.Increased or decreased expression of a BASB006 polynucleotide can bemeasured using any on of the methods well known in the art for thequantitation of polynucleotides, such as, for example, amplification.PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and otherhybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of BASB006 polypeptide compared to normalcontrol tissue samples may be used to detect the presence of aninfection, for example. Assay techniques that can be used to determinelevels of a BASB006 polypeptide, in a sample derived from a host, suchas a bodily material, are well-known to those of skill in the art. Suchassay methods include radioimmunoassays, competitive-binding assays,Western Blot analysis, antibody sandwich assays, antibody detection andELISA assays.

The polynucleotides of the invention may be used as components ofpolynucleotide arrays, preferably high density arrays or grids. Thesehigh density arrays are particularly useful for diagnostic andprognostic purposes. For example, a set of spots each comprising adifferent gene, and further comprising a polynucleotide orpolynucleotides of the invention, may be used for probing, such as usinghybridization or nucleic acid amplification, using a probe obtained orderived from a bodily sample, to determine the presence of a particularpolynucleotide sequence or related sequence in an individual. Such apresence may indicate the presence of a pathogen, particularly Neisseriameningitidis, and may be useful in diagnosing and/or prognosing diseaseor a course of disease. A grid comprising a number of variants of thepolynucleotide sequence of SEQ ID NO:1, 3 are preferred. Also preferredis a grid comprising a number of variants of a polynucleotide sequenceencoding the polypeptide sequence of SEQ ID NO:2, 4.

Antibodies

The polypeptides and polynucleotides of the invention or variantsthereof, or cells expressing the same can be used as immunogens toproduce antibodies immunospecific for such polypeptides orpolynucleotides respectively.

In certain preferred embodiments of the invention there are providedantibodies against BASB006 polypeptides or polynucleotides.

Antibodies generated against the polypeptides or polynucleotides of theinvention can be obtained by administering the polypeptides and/orpolynucleotides of the invention, or epitope-bearing fragments of eitheror both, analogues of either or both, or cells expressing either orboth, to an animal, preferably a nonhuman, using routine protocols. Forpreparation of monoclonal antibodies, any technique known in the artthat provides antibodies produced by continuous cell line cultures canbe used. Examples include various techniques, such as those in Kohler,G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides or polynucleotides of this invention. Also, transgenicmice, or other organisms or animals, such as other mammals. may be usedto express humanized antibodies immunospecific to the polypeptides orpolynucleotides of the invention.

Alternatively, phage display technology may be utilized to selectantibody genes with binding activities towards a polypeptide of theinvention either from repertoires of PCR amplified v-genes oflymphocytes from humans screened for possessing anti-BASB006 or fromnaive libraries (McCafferty, et al., (1990), Nature 348 552-554; Marks.et al., (1992) Biotechnology 10, 779-783). The affinity of theseantibodies can also be improved by, for example, chain shuffling(Clackson et al., (1991) Nature 352: 628).

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides or polynucleotides of the inventionto purify the polypeptides or polynucleotides by, for example, affinitychromatography.

Thus, among others, antibodies against BASB006-polypeptide orBASB006-polynucleotide may be employed to treat infections, particularlybacterial infections.

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants form a particular aspect of thisinvention.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be “humanized,” where thecomplimentarity determining region or regions of the hybridoma-derivedantibody has been transplanted into a human monoclonal antibody, forexample as described in Jones et al. (1986), Nature 321, 522-525 orTempest et al., (1991) Biotechnology 9, 266-273.

Antagonists and Agonists—Assays and Molecules

Polypeptides and polynucleotides of the invention may also be used toassess the binding of small molecule substrates and ligands in, forexample, cells, cell-free preparations, chemical libraries, and naturalproduct mixtures. These substrates and ligands may be natural substratesand ligands or may be structural or functional mimetics. See, e.g,Coligan et al., Current Protocols in Immunology 1(2). Chapter 5 (1991).

The screening methods may simply measure the binding of a candidatecompound to the polypeptide or polynucleotide, or to cells or membranesbearing the polypeptide or polynucleotide, or a fusion protein of thepolypeptide by means of a label directly or indirectly associated withthe candidate compound. Alternatively, the screening method may involvecompetition with a labeled competitor. Further, these screening methodsmay test whether the candidate compound results in a signal generated byactivation or inhibition of the polypeptide or polynucleotide, usingdetection systems appropriate to the cells comprising the polypeptide orpolynucleotide. Inhibitors of activation are generally assayed in thepresence of a known agonist and the effect on activation by the agonistby the presence of the candidate compound is observed. Constitutivelyactive polypeptide and/or constitutively expressed polypeptides andpolynucleotides may be employed in screening methods for inverseagonists or inhibitors, in the absence of an agonist or inhibitor, bytesting whether the candidate compound results in inhibition ofactivation of the polypeptide or polynucleotide, as the case may be.Further, the screening methods may simply comprise the steps of mixing acandidate compound with a solution containing a polypeptide orpolynucleotide of the present invention, to form a mixture, measuringBASB006 polypeptide and/or polynucleotide activity in the mixture, andcomparing the BASB006 polypeptide and/or polynucleotide activity of themixture to a standard. Fusion proteins, such as those made from Fcportion and BASB006 polypeptide, as hereinbefore described, can also beused for high-throughput screening assays to identify antagonists of thepolypeptide of the present invention, as well as of phylogenetically andand/or functionally related polypeptides (see D. Bennett et al., J MolRecognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem,270(16):9459-9471 (1995)).

The polynucleotides, polypeptides and antibodies that bind to and/orinteract with a polypeptide of the present invention may also be used toconfigure screening methods for detecting the effect of added compoundson the production of mRNA and/or polypeptide in cells. For example, anELISA assay may be constructed for measuring, secreted or cellassociated levels of polypeptide using monoclonal and polyclonalantibodies by standard methods known in the art. This can be used todiscover agents which may inhibit or enhance the production ofpolypeptide (also called antagonist or agonist, respectively) fromsuitably manipulated cells or tissues.

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action ofBASB006 polypeptides or polynucleotides, particularly those compoundsthat are bacteristatic and/or bactericidal. The method of screening mayinvolve high-throughput techniques. For example, to screen for agonistsor antagonists, a synthetic reaction mix, a cellular compartment, suchas a membrane, cell envelope or cell wall, or a preparation of anythereof, comprising BASB006 polypeptide and a labeled substrate orligand of such polypeptide is incubated in the absence or the presenceof a candidate molecule that may be a BASB006 agonist or antagonist. Theability of the candidate molecule to agonize or antagonize the BASB006polypeptide is reflected in decreased binding of the labeled ligand ordecreased production of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of BASB006 polypeptideare most likely to be good antagonists. Molecules that bind well and, asthe case may be, increase the rate of product production from substrate,increase signal transduction, or increase chemical channel activity areagonists. Detection of the rate or level of, as the case may be,production of product from substrate, signal transduction, or chemicalchannel activity may be enhanced by using a reporter system. Reportersystems that may be useful in this regard include but are not limited tocalorimetric labeled substrate converted into product, a reporter genethat is responsive to changes in BASB006 polynucleotide or polypeptideactivity, and binding assays known in the art.

Another example of an assay for BASB006 agonists is a competitive assaythat combines BASB006 and a potential agonist with BASB006-bindingmolecules, recombinant BASB006 binding molecules, natural substrates orligands, or substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. BASB006 can be labeled, such as byradioactivity or a colorimetric compound, such that the number ofBASB006 molecules bound to a binding molecule or converted to productcan be determined accurately to assess the effectiveness of thepotential antagonist.

Potential antagonists include, among others, small organic molecules,peptides, polypeptides and antibodies that bind to a polynucleotideand/or polypeptide of the invention and thereby inhibit or extinguishits activity or expression. Potential antagonists also may be smallorganic molecules, a peptide, a polypeptide such as a closely relatedprotein or antibody that binds the same sites on a binding molecule,such as a binding molecule, without inducing BASB006-induced activities,thereby preventing the action or expression of BASB006 polypeptidesand/or polynucleotides by excluding BASB006 polypeptides and/orpolynucleotides from binding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56:560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of BASB006.

In a further aspect, the present invention relates to geneticallyengineered soluble fusion proteins comprising a polypeptide of thepresent invention, or a fragment thereof, and various portions of theconstant regions of heavy or light chains of immunoglobulins of varioussubclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is theconstant part of the heavy chain of human IgG, particularly IgG1, wherefusion takes place at the hinge region. In a particular embodiment, theFc part can be removed simply by incorporation of a cleavage sequencewhich can be cleaved with blood clotting factor Xa. Furthermore, thisinvention relates to processes for the preparation of these fusionproteins by genetic engineering and to the use thereof for drugscreening diagnosis and therapy. A further aspect of the invention alsorelates to polynucleotides encoding such fusion proteins. Examples offusion protein technology can be found in International PatentApplication Nos. WO94/29458 and WO94/22914.

Each of the polynucleotide sequences provided herein may be used in thediscovery and development of antibacterial compounds. The encodedprotein, upon expression, can be used as a target for the screening ofantibacterial drugs. Additionally, the polynucleotide sequences encodingthe amino terminal regions of the encoded protein or Shine-Delgarno orother translation facilitating sequences of the respective mRNA can beused to construct antisense sequences to control the expression of thecoding sequence of interest.

The invention also provides the use of the polypeptide, polynucleotide,agonist or antagonist of the invention to interfere with the initialphysical interaction between a pathogen or pathogens and a eukaryotic,preferably mammalian, host responsible for sequelae of infection. Inparticular, the molecules of the invention may be used: in theprevention of adhesion of bacteria, in particular gram positive and/orgram negative bacteria, to eukaryotic, preferably mammalian,extracellular matrix proteins on in-dwelling devices or to extracellularmatrix proteins in wounds, to block bacterial adhesion betweeneukaryotic, preferably mammalian, extracellular matrix proteins andbacterial BASB006 proteins that mediate tissue damage and/or; to blockthe normal progression of pathogenesis in infections initiated otherthan by the implantation of in-dwelling devices or by other surgicaltechniques.

In accordance with yet another aspect of the invention, there areprovided BASB006 agonists and antagonists, preferably bacteristatic orbactericidal agonists and antagonists.

The antagonists and agonists of the invention may be employed, forinstance, to prevent, inhibit and/or treat diseases.

In a further aspect, the present invention relates to mimotopes of thepolypeptide of the invention. A mimotope is a peptide sequence,sufficiently similar to the native peptide (sequentially orstructurally), which is capable of being recognised by antibodies whichrecognise the native peptide; or is capable of raising antibodies whichrecognise the native peptide when coupled to a suitable carrier.

Peptide mimotopes may be designed for a particular purpose by addition,deletion or substitution of elected amino acids. Thus, the peptides maybe modified for the purposes of ease of conjugation to a proteincarrier. For example, it may be desirable for some chemical conjugationmethods to include a terminal cysteine. In addition it may be desirablefor peptides conjugated to a protein carrier to include a hydrophobicterminus distal from the conjugated terminus of the peptide, such thatthe free unconjugated end of the peptide remains associated with thesurface of the carrier protein. Thereby presenting the peptide in aconformation which most closely resembles that of the peptide as foundin the context of the whole native molecule. For example, the peptidesmay be altered to have an N-terminal cysteine and a C-terminalhydrophobic amidated tail. Alternatively, the addition or substitutionof a D-stereoisomer form of one or more of the amino acids may beperformed to create a beneficial derivative, for example to enhancestability of the peptide.

Alternatively, peptide mimotopes may be identified using antibodieswhich are capable themselves of binding to the polypeptides of thepresent invention using techniques such as phage display technology (EP0 552 267 B1). This technique, generates a large number of peptidesequences which mimic the structure of the native peptides and are,therefore, capable of binding to anti-native peptide antibodies, but maynot necessarily themselves share significant sequence homology to thenative polypeptide.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal,preferably humans, which comprises inoculating the individual withBASB006 polynucleotide and/or polypeptide, or a fragment or variantthereof, adequate to produce antibody and/ or T cell immune response toprotect said individual from infection, particularly bacterial infectionand most particularly Neisseria meningitidis infection. Also providedare methods whereby such immunological response slows bacterialreplication. Yet another aspect of the invention relates to a method ofinducing immunological response in an individual which comprisesdelivering to such individual a nucleic acid vector, sequence orribozyme to direct expression of BASB006 polynucleotide and/orpolypeptide, or a fragment or a variant thereof, for expressing BASB006polynucleotide and/or polypeptide, or a fragment or a variant thereof invivo in order to induce an immunological response, such as, to produceantibody and/ or T cell immune response, including, for example,cytokine-producing T cells or cytotoxic T cells, to protect saidindividual, preferably a human, from disease, whether that disease isalready established within the individual or not. One example ofadministering the gene is by accelerating it into the desired cells as acoating on particles or otherwise. Such nucleic acid vector may compriseDNA, RNA, a ribozyme, a modified nucleic acid, a DNA/RNA hybrid, aDNA-protein complex or an RNA-protein complex.

A further aspect of the invention relates to an immunologicalcomposition that when introduced into an individual, preferably a human,capable of having induced within it an immunological response, inducesan immunological response in such individual to a BASB006 polynucleotideand/or polypeptide encoded therefrom, wherein the composition comprisesa recombinant BASB006 polynucleotide and/or polypeptide encodedtherefrom and/or comprises DNA and/or RNA which encodes and expresses anantigen of said BASB006 polynucleotide, polypeptide encoded therefrom,or other polypeptide of the invention. The immunological response may beused therapeutically or prophylactically and may take the form ofantibody immunity and/or cellular immunity, such as cellular immunityarising from CTL or CD4+T cells.

A BASB006 polypeptide or a fragment thereof may be fused with co-proteinor chemical moiety which may or may not by itself produce antibodies,but which is capable of stabilizing the first protein and producing afused or modified protein which will have antigenic and/or immunogenicproperties, and preferably protective properties. Thus fused recombinantprotein, preferably further comprises an antigenic co-protein, such aslipoprotein D from Haemophilus influenzae, Glutathione-S-transferase(GST) or beta-galactosidase, or any other relatively large co-proteinwhich solubilizes the protein and facilitates production andpurification thereof. Moreover, the co-protein may act as an adjuvant inthe sense of providing a generalized stimulation of the immune system ofthe organism receiving the protein. The co-protein may be attached toeither the amino- or carboxy-terminus of the first protein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides and/orpolynucleotides of the invention and immunostimulatory DNA sequences,such as those described in Sato, Y. et al. Science 273:352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins, inpolynucleotide constructs used in such genetic immunization experimentsin animal models of infection with Neisseria meningitidis. Suchexperiments will be particularly useful for identifying protein epitopesable to provoke a prophylactic or therapeutic immune response. It isbelieved that this approach will allow for the subsequent preparation ofmonoclonal antibodies of particular value, derived from the requisiteorgan of the animal successfully resisting or clearing infection, forthe development of prophylactic agents or therapeutic treatments ofbacterial infection, particularly Neisseria meningitidis infection, inmammals, particularly humans.

The invention also includes a vaccine formulation which comprises animmunogenic recombinant polypeptide and/or polynucleotide of theinvention together with a suitable carrier, such as a pharmaceuticallyacceptable carrier. Since the polypeptides and polynucleotides may bebroken down in the stomach, each is preferably administeredparenterally, including, for example, administration that issubcutaneous, intramuscular, intravenous, or intradermal. Formulationssuitable for parenteral administration include aqueous and non-aqueoussterile injection solutions which may contain anti-oxidants, buffers,bacteristatic compounds and solutes which render the formulationisotonic with the bodily fluid, preferably the blood, of the individual;and aqueous and non-aqueous sterile suspensions which may includesuspending agents or thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampoules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use.

The vaccine formulation of the invention may also include adjuvantsystems for enhancing the immunogenicity of the formulation. Preferablythe adjuvant system raises preferentially a TH1 type of response.

An immune response may be broadly distinguished into two extremecatagories, being a humoral or cell mediated immune responses(traditionally characterised by antibody and cellular effectormechanisms of protection respectively). These categories of responsehave been termed TH1-type responses (cell-mediated response), andTH2-type immune responses (humoral response).

Extreme TH1-type immune responses may be characterised by the generationof antigen specific, haplotype restricted cytotoxic T lymphocytes, andnatural killer cell responses. In mice TH1-type responses are oftencharacterised by the generation of antibodies of the IgG2a subtype,whilst in the human these correspond to IgG1 type antibodies. TH2-typeimmune responses are characterised by the generation of a broad range ofimmunoglobulin isotypes including in mice IgG1, IgA, and IgM.

It can be considered that the driving force behind the development ofthese two types of immune responses are cytokines. High levels ofTH1-type cytokines tend to favour the induction of cell mediated immuneresponses to the given antigen, whilst high levels of TH2-type cytokinestend to favour the induction of humoral immune responses to the antigen.

The distinction of TH1 and TH2-type immune responses is not absolute. Inreality an individual will support an immune response which is describedas being predominantly TH1 or predominantly TH2. However, it is oftenconvenient to consider the families of cytokines in terms of thatdescribed in murine CD4+ve T cell clones by Mosmann and Coffman(Mosonzann, T. R. and Coffman, R. L. (1989) TH1and TH2 cells: differentpatterns of lymphokine secretion lead to different functionalproperties. Annual Review of Immunology, 7, p145-173). Traditionally,TH1-type responses are associated with the production of the INF-γ andIL-2 cytokines by T-lymphocytes. Other cytokines often directlyassociated with the induction of TH1-type immune responses are notproduced by T-cells, such as IL-12. In contrast, TH2- type responses areassociated with the secretion of IL-4, IL-5, IL-6 and IL-I 13.

It is known that certain vaccine adjuvants are particularly suited tothe stimulation of either TH1 or TH2- type cytokine responses.Traditionally the best indicators of the TH1 :TH2 balance of the immuneresponse after a vaccination or infection includes direct measurement ofthe production of TH1 or TH2 cytokines by T lymphocytes in vitro afterrestimulation with antigen, and/or the measurement of the IgG1:IgG2aratio of antigen specific antibody responses.

Thus, a TH1-type adjuvant is one which preferentially stimulatesisolated T-cell populations to produce high levels of TH1-type cytokineswhen re-stimulated with antigen in vitro, and promotes development ofboth CD8+ cytotoxic T lymphocytes and antigen specific immunoglobulinresponses associated with TH1-type isotype.

Adjuvants which are capable of preferential stimulation of the TH1 cellresponse are described in International Patent Application No. WO94/00153 and WO 95/17209.

3 De-O-acylated monophosphoryl lipid A (3D-MPL) is one such adjuvant.This is known from GB 2220211 (Ribi). Chemically it is a mixture of 3De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains andis manufactured by Ribi

Immunochem, Montana. A preferred form of 3 De-O-acylated monophosphoryllipid A is disclosed in European Patent 0 689 454 B1 (SmithKline BeechamBiologicals SA).

Preferably, the particles of 3D-MPL are small enough to be sterilefiltered through a 0.22 micron membrane (European Patent number 0 689454). 3D-MPL will be present in the range of 10 μg-100 μg preferably25-50 μg per dose wherein the antigen will typically be present in arange 2-50 μg per dose.

Another preferred adjuvant comprises QS21, an Hplc purified non-toxicfraction derived from the bark of Quillaja Saponaria Molina. Optionallythis may be admixed with 3 De-O-acylated monophosphoryl lipid A(3D-MPL), optionally together with a carrier.

The method of production of QS21 is disclosed in U.S. Pat. No.5,057,540.

Non-reactogenic adjuvant formulations containing QS21 have beendescribed previously (WO 96/33739). Such formulations comprising QS21and cholesterol have been shown to be successful TH1 stimulatingadjuvants when formulated together with an antigen.

Further adjuvants which are preferential stimulators of TH1 cellresponse include immunomodulatory oligonucleotides, for exampleunmethylated CpG sequences as disclosed in WO 96/02555.

Combinations of different TH1 stimulating adjuvants, such as thosementioned hereinabove, are also contemplated as providing an adjuvantwhich is a preferential stimulator of TH1 cell response. For example,QS21 can be formulated together with 3D-MPL. The ratio of QS21:3D-MPLwill typically be in the order of 1:10 to 10:1; preferably 1:5 to 5:1and often substantially 1:1. The preferred range for optimal synergy is2.5:1 to 1:13 D-MPL: QS21.

Preferably a carrier is also present in the vaccine compositionaccording to the invention. The carrier may be an oil in water emulsion,or an aluminium salt, such as aluminium phosphate or aluminiumhydroxide.

A preferred oil-in-water emulsion comprises a metabolisible oil, such assqualene, alpha tocopherol and Tween 80. In a particularly preferredaspect the antigens in the vaccine composition according to theinvention are combined with QS21 and 3D-MPL in such an emulsion.Additionally the oil in water emulsion may contain span 85 and/orlecithin and/or tricaprylin.

Typically for human administration QS21 and 3D-MPL will be present in avaccine in the range of 1 μg-200 μg, such as 10-100 μg, preferably 10μg-50 μg per dose. Typically the oil in water will comprise from 2 to10% squalene, from 2 to 10% alpha tocopherol and from 0.3 to 3% tween80. Preferably the ratio of squalene: alpha tocopherol is equal to orless than 1 as this provides a more stable emulsion. Span 85 may also bepresent at a level of 1%. In some cases it may be advantageous that thevaccines of the present invention will further contain a stabiliser.

Non-toxic oil in water emulsions preferably contain a non-toxic oil,e.g. squalane or squalene, an emulsifier, e.g. Tween 80, in an aqueouscarrier. The aqueous carrier may be, for example, phosphate bufferedsaline.

A particularly potent adjuvant formulation involving QS21, 3D-MPL andtocopherol in an oil in water emulsion is described in WO 95/17210.

The present invention also provides a polyvalent vaccine compositioncomprising a vaccine formulation of the invention in combination withother antigens, in particular antigens useful for treating cancers,autoimmune diseases and related conditions. Such a polyvalent vaccinecomposition may include a TH-1 inducing adjuvant as hereinbeforedescribed.

While the invention has been described with reference to certain BASB006polypeptides and polynucleotides, it is to be understood that thiscovers fragments of the naturally occurring polypeptides andpolynucleotides, and similar polypeptides and polynucleotides withadditions, deletions or substitutions which do not substantially affectthe immunogenic properties of the recombinant polypeptides orpolynucleotides.

The antigen can also be delivered in the form of whole bacteria (dead oralive) or as subcellular fractions, these possibilities do include N.meningitidis itself.

Compositions, Kits and Administration

In a further aspect of the invention there are provided compositionscomprising a BASB006 polynucleotide and/or a BASB006 polypeptide foradministration to a cell or to a multicellular organism.

The invention also relates to compositions comprising a polynucleotideand/or a polypeptide discussed herein or their agonists or antagonists.The polypeptides and polynucleotides of the invention may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to an individual. Such compositionscomprise, for instance, a media additive or a therapeutically effectiveamount of a polypeptide and/or polynucleotide of the invention and apharmaceutically acceptable carrier or excipient. Such carriers mayinclude, but are not limited to, saline, buffered saline, dextrose,water, glycerol, ethanol and combinations thereof. The formulationshould suit the mode of administration. The invention further relates todiagnostic and pharmaceutical packs and kits comprising one or morecontainers filled with one or more of the ingredients of theaforementioned compositions of the invention.

Polypeptides, polynucleotides and other compounds of the invention maybe employed alone or in conjunction with other compounds, such astherapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

In a further aspect, the present invention provides for pharmaceuticalcompositions comprising a therapeutically effective amount of apolypeptide and/or polynucleotide, such as the soluble form of apolypeptide and/or polynucleotide of the present invention, agonist orantagonist peptide or small molecule compound, in combination with apharmaceutically acceptable carrier or excipient. Such carriers include,but are not limited to, saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The invention furtherrelates to pharmaceutical packs and kits comprising one or morecontainers filled with one or more of the ingredients of theaforementioned compositions of the invention. Polypeptides,polynucleotides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

The composition will be adapted to the route of administration, forinstance by a systemic or an oral route. Preferred forms of systemicadministration include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if a polypeptide or other compounds of the present inventioncan be formulated in an enteric or an encapsulated formulation, oraladministration may also be possible. Administration of these compoundsmay also be topical and/or localized, in the form of salves, pastes,gels, solutions, powders and the like.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

The dosage range required depends on the choice of peptide, the route ofadministration, the nature of the formulation, the nature of thesubject's condition, and the judgment of the attending practitioner.Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Wide variations in the needed dosage, however, are to be expected inview of the variety of compounds available and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization, as is well understood in the art.

Sequence Databases, Sequences in a Tangible Medium, and Algorithms

Polynucleotide and polypeptide sequences form a valuable informationresource with which to determine their 2- and 3-dimensional structuresas well as to identify further sequences of similar homology. Theseapproaches are most easily facilitated by storing the sequence in acomputer readable medium and then using the stored data in a knownmacromolecular structure program or to search a sequence database usingwell known searching tools, such as the GCG program package.

Also provided by the invention are methods for the analysis of charactersequences or strings, particularly genetic sequences or encoded proteinsequences. Preferred methods of sequence analysis include, for example,methods of sequence homology analysis, such as identity and similarityanalysis, DNA, RNA and protein structure analysis, sequence assembly,cladistic analysis, sequence motif analysis, open reading framedetermination, nucleic acid base calling, codon usage analysis, nucleicacid base trimming, and sequencing chromatogram peak analysis.

A computer based method is provided for performing homologyidentification. This method comprises the steps of: providing a firstpolynucleotide sequence comprising the sequence of a polynucleotide ofthe invention in a computer readable medium; and comparing said firstpolynucleotide sequence to at least one second polynucleotide orpolypeptide sequence to identify homology.

A computer based method is also provided for performing homologyidentification, said method comprising the steps of: providing a firstpolypeptide sequence comprising the sequence of a polypeptide of theinvention in a computer readable medium; and comparing said firstpolypeptide sequence to at least one second polynucleotide orpolypeptide sequence to identify homology.

All publications and references, including but not limited to patentsand patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

DEFINITIONS

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, as thecase may be, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods, including but not limited tothose described in (Computational Mololecular, Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey. 1994; Sequence Analysis inMolecular Biology, von Heine, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press.New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math.,48: 1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computer programs.Computer program methods to determine identity between two sequencesinclude, but are not limited to the GAP program in the GCG programpackage (Devereux, J., et al., Nucleic Acids Research 12(1): 387(1984)), BLASTP, BLASTN (Altschul, S. F. et al., .J. Molec. Biol. 215:403-410 (1990), and FASTA( Pearson and Lipman Proc. Natl. Acad. Sci. USA85; 2444-2448 (1988). The BLAST family of programs is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Miol. Biol. 215:403-410 (1990). The well known Smith Waterman algorithm may also be usedto determine identity.

Parameters for polypeptide sequence comparison include the following:

Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: BLOSSUM62 from Henikoff and Henikoff,

Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992)

Gap Penalty: 8

Gap Length Penalty: 2

A program useful with these parameters is publicly available as the“gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

Parameters for polynucleotide comparison include the following:

Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

Available as: The “gap” program from Genetics Computer Group, MadisonWis. These are the default parameters for nucleic acid comparisons.

A preferred meaning for “identity” for polynucleotides and polypeptides,as the case may be, are provided in (1) and (2) below.

(1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the referencesequence of SEQ ID NO:1, wherein said polynucleotide sequence may beidentical to the reference sequence of SEQ ID NO:1 or may include up toa certain integer number of nucleotide alterations as compared to thereference sequence, wherein said alterations are selected from the groupconsisting of at least one nucleotide deletion, substitution, includingtransition and transversion, or insertion, and wherein said alterationsmay occur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among the nucleotides in the reference sequence orin one or more contiguous groups within the reference sequence, andwherein said number of nucleotide alterations is determined bymultiplying the total number of nucleotides in SEQ ID NO:1 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of nucleotides in SEQ IDNO:1, or:

n _(n) ≦x _(n)−(x _(n) ·y)

wherein n_(n) is the number of nucleotide alterations, x_(n) is thetotal number of nucleotides in SEQ ID NO:1, y is 0.50 for 50%, 0.60 for60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for95%, 0.97 for 97% or 1.00 for 100%, and · is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n). Alterations of a polynucleotide sequence encoding thepolypeptide of SEQ ID NO:2 may create nonsense, missense or frameshiftmutations in this coding sequence and thereby alter the polypeptideencoded by the polynucleotide following such alterations.

By way of example, a polynucleotide sequence of the present inventionmay be identical to the reference sequence of SEQ ID NO:1, that is itmay be 100% identical, or it may include up to a certain integer numberof nucleic acid alterations as compared to the reference sequence suchthat the percent identity is less than 100% identity. Such alterationsare selected from the group consisting of at least one nucleic aciddeletion substitution, including transition and transversion, orinsertion, and wherein said alterations may occur at the 5′ or 3′terminal positions of the reference polynucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongthe nucleic acids in the reference sequence or in one or more contiguousgroups within the reference sequence. The number of nucleic acidalterations for a given percent identity is determined by multiplyingthe total number of nucleic acids in SEQ ID NO:1 by the integer definingthe percent identity divided by 100 and then subtracting that productfrom said total number of nucleic acids in SEQ ID NO:1, or:

n _(n) ≦x _(n)−(x _(n) ·y),

wherein n_(n) is the number of nucleic acid alterations, x_(n) is thetotal number of nucleic acids in SEQ ID NO:1, y is, for instance 0.70for 70%, 0.80 for 80%, 0.85 for 85% etc., · is the symbol for themultiplication operator, and wherein any non-integer product of X_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n).

(2) Polypeptide embodiments further include an isolated polypeptidecomprising a polypeptide having at least a 50, 60, 70, 80, 85, 90, 95,97 or 100% identity to a polypeptide reference sequence of SEQ ID NO:2,wherein said polypeptide sequence may be identical to the referencesequence of SEQ ID NO:2 or may include up to a certain integer number ofamino acid alterations as compared to the reference sequence, whereinsaid alterations are selected from the group consisting of at least oneamino acid deletion, substitution, including conservative andnon-conservative substitution, or insertion, and wherein saidalterations may occur at the amino- or carboxy-terminal positions of thereference polypeptide sequence or anywhere between those terminalpositions, interspersed either individually among the amino acids in thereference sequence or in one or more contiguous groups within thereference sequence, and wherein said number of amino acid alterations isdetermined by multiplying the total number of amino acids in SEQ ID NO:2by the integer defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

n _(a) ≦x _(a)−(x _(a) ·y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is 0.50 for 50%, 0.60 for60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for95%, 0.97 for 97% or 1.00 for 100%, and · is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

By way of example, a polypeptide sequence of the present invention maybe identical to the reference sequence of SEQ ID NO:2, that is it may be100% identical, or it may include up to a certain integer number ofamino acid alterations as compared to the reference sequence such thatthe percent identity is less than 100% identity. Such alterations areselected from the group consisting of at least one amino acid deletion.substitution, including conservative and non-conservative substitution,or insertion, and wherein said alterations may occur at the amino- orcarboxy-terninal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofamino acid alterations for a given % identity is determined bymultiplying the total number of amino acids in SEQ ID NO:2 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

n _(a) ≦x _(a)−(x _(a) ·y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is, for instance 0.70 for70%, 0.80 for 80%, 0.85 for 85% etc., and · is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

“Individual(s),” when used herein with reference to an organism, means amulticellular eukaryote, including, but not limited to a metazoan, amammal, an ovid, a bovid, a simian, a primate, and a human.

“Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is “isolated” even if it is still present in saidorganism, which organism may be living or non-living.

“Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA including single and double-stranded regions.

“Variant” refers to a polynucleotide or polypeptide that differs from areference polynucleotide or polypeptide, but retains essentialproperties. A typical variant of a polynucleotide differs in nucleotidesequence from another, reference polynucleotide. Changes in thenucleotide sequence of the variant may or may not alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence, as discussed below. A typical variant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additions,deletions in any combination. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. A variant ofa polynucleotide or polypeptide may be a naturally occurring such as anallelic variant, or it may be a variant that is not known to occurnaturally. Non-naturally occurring variants of polynucleotides andpolypeptides may be made by mutagenesis techniques or by directsynthesis.

“Disease(s)” means any disease caused by or related to infection by abacteria, including, for example, upper respiratory tract infection,invasive bacterial diseases, such as bacteremia and meningitis.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1 Discovery and Confirmatory DNA Sequencing of the BASB006 Genefrom two N. meninigitidis Strains

A: BASB006 in N. meningitidis serogroup B strain ATCC13090.

The BASB006 gene disclosed in SEQ ID NO:1 was first discovered in theIncyte PathoSeq database containing unfinished genomic DNA sequences ofthe N. meningitidis strain ATCCI13090. The translation of the BASB006polynucleotide sequence, shown in SEQ ID NO:2, showed significantsimilarity (56% identity in a 1455 amino acids overlap) to the Happrotein of Haemophilis influenzae, a polypeptide reported previously tofunction as an adhesin. The sequence of the BASB006 gene was confirmedexperimentally. For this purpose, genomic DNA was extracted from 10¹⁰cells of the N.meningitidis cells (strain ATCC 13090) using the QIAGENgenomic DNA extraction kit (Qiagen Gmbh), and 1 μg of this material wassubmitted to Polymerase Chain Reaction DNA amplification using primersHap01 (5′-GGG GGC TAG CAA AAC AAC CGA CAA ACG GAC AAC C-3′) [SEQ IDNO:5] and Hap02 (5′-GGG GAA GCT TCC AGC GGT AGC GGT AGC CTA ATT TGA TGCC-3′) [SEQ ID NO:6]. This PCR product was gel-purified and subjected toDNA sequencing using the Big Dye Cycle Sequencing kit (Perkin-Elmer) andan ABI 373A/PRISM DNA sequencer. DNA sequencing was performed on bothstrands with a redundancy of 2 and the full-length sequence wasassembled using the SeqMan program from the DNASTAR Lasergene softwarepackage The resulting DNA sequence turned out to be 100 % identical toSEQ ID NO:1.

B: BASB006 in N. meningitidis serogroup B strain H44/76.

The sequence of the BASB006 gene was also determined in another N.meningitidis serogroup B strain, the strain H144/76. For this purpose,genomic DNA was extracted from the N. meningitidis strain H44/76 usingthe experimental conditions presented in Example 1. This material (1 μg)was then submitted to Polymerase Chain Reaction DNA amplification usingprimers Hap01 and Hap02 specific for the BASB006 gene. A 4389bp DNAfragment was obtained, digested by the NheI/HindIII restrictionendonucleases and inserted into the corresponding sites of the pET-24bcloning/expression vector (Novagen) using standard molecular biologytechniques (Molecular Cloning,a Laboratory Manual, Second Edition, Eds:Sambrook, Fritsch & Maniatis, Cold Spring Harbor press 1989).Recombinant pET-24b/BASB006 was then submitted to DNA sequencing usingthe Big Dyes kit (Applied biosystems) and analyzed on a ABI 373/A DNAsequencer in the conditions described by the supplier. As a result, thepolynucleotide and deduced polypeptide sequences, referred to as SEQ IDNO:3 and SEQ ID NO:4 respectively, were obtained. Using the PILEUPprogram from the GCG package, an alignment of the polynucleotidesequences of SEQ ID NO:1 and 3 was performed, and is displayed in FIG.1; their level of identity amounts to 97.8 %, as determined by the GAPprogram. Using the same PILEUP program, an alignment of the polypeptidesequences of SEQ ID NO:2 and 4 was performed, and is displayed in FIG.2; their level of identity amounts to 97.0%, as determined by the GAPprogram.

Taken together, these data indicate strong sequence conservation of theBASB006 gene among the two N.meningitidis serogroup B strains.

Example 2 Expression and Purification of Recombinant BASB006 Protein inEscherichia coli

The construction of the pET-24b/BASB006 cloning/expression vector wasdescribed in Example 1B. This vector harbours the BASB006 gene isolatedfrom the strain H44/76 in fusion with a stretch of 6 histidine residues,placed under the control of the strong bacteriophage T7 gene 10promoter. For expression study, this vector was introduced into theEscherichia coli strain BL21 DE3 (Novagen), in which, the gene for theT7 polymerase is placed under the control of the isopropyl-beta-Dthiogalactoside (IPTG)-regulatable lac promoter. Liquid cultures (100ml) of the BL21 DE-3 [pET-24b/BASB006] E. coli recombinant strain weregrown at 37° C. under agitation until the optical density at 600 nm(OD600) reached 0.6. At that time-point, IPTG was added at a finalconcentration of 1 mM and the culture was grown for 4 additional hours.The culture was then centrifuged at 10,000 rpm and the pellet was frozenat −20° C. for at least 10 hours. After thawing, the pellet wasresuspended during 30 min at 25° C. in buffer A (6M guanidinehydrochloride, 0.1M NaH2PO4, 0.01M Tris, pH 8.0), passed three-timesthrough a needle and clarified by centrifugation (20000 rpm, 15 min).The sample was then loaded at a flow-rate of 1 ml/min on a Ni2+-loadedHitrap column (Pharmacia Biotech). After passsage of the flowthrough,the column was washed succesively with 40 ml of buffer B (8M Urea, 0.1MNaH2PO4, 0.01M Tris, pH 8.0), 40 ml of buffer C (8M Urea, 0.1M NaH2PO4,0.01M Tris, pH 6.3). The recombinant protein BASB006/His6 was theneluted from the column with 30 ml of buffer C (8M Urea, 0.1 M NaH2PO4,0.01M Tris, pH 6.3) containing 500 mM of imidazole and 3 ml-sizefractions were collected. In FIG. 3 substantially purified proteins wereseparated on a 4-20% gradient polyacrylamide gel under PAGE-SDSconditions and stained with Coomassie Blue R250. The sample loaded onthe gel corresponded to protein fractions enriched (more than 80%) inBASB006 (lane 1 and 2) and a molecular weight marker (MW). As shown inFIG. 3, a highly enriched (Purity estimated to more than 90% pure incoomassie staining) BASB006/His6 protein, migrating at 170 kDa(estimated relative molecular mass), was eluted from the column. Thispolypeptide was reactive against a mouse monoclonal antibody raisedagainst the 5-histidine motif. Taken together, these data indicate thatthe BASB006 gene can be expressed and purified under a recombinant form(BASB006/His6) in E.coli.

Example 3 Immunization of Mice with Recombinant BASB006 and Recognitionof the BASB006 Polypeptide on Different N. meningitidis Serogroup BStrains by Western Blotting.

Partially purified recombinant BASB006 expressed in E. coli has beeninjected three times in BALB/C mice on days 0, 14 and 28 (10animals/group). Animals were injected by the subcutaneous route with 5μg of antigen r formulated in SB62 emulsion containing 5 μg MPL, and 1μpg QS21 per dose. Mice were bled on days 29 (15 days Post II) and 35 (6days Post III) in order to detect specific anti-BASB006 antibodies.Specific anti-BASB006 antibodies were measured on pooled sera (from 10mice/group) by western-blotting on six different Neisseria meningitidisserogroup B strains (FIG. 4).

The six different Neisseria meningitidis B strains are: H44/76(B:15:P1.7, 16, lineage ET-5), M97 250987 (B:4:P1.15), BZ10 (B:2b:P1.2,lineage A4), BZ198 (B:NT*:-, lineage 3), and EG328 (B:NT*, lineageST-18), and on partially purified recombinant BASB006 protein (mixedwith two other candidate antigens). (*: NT: Not Typed).

Briefly, 15 μl (>10⁸ cells/lane) of each sample treated with samplebuffer (10 min at 95° C.) are put into a SDS-PAGE gradient gel(Tris-glycine 4-20%, Novex, code n°EC60252). Electrophoretic migrationoccurs at 125 volts for 90 min. Afterwards, proteins are transferred toa nitrocellulose sheet (0.45 μm, Bio-rad code n° 162-0114) at 100 voltsfor 1 hour using a Bio-rad Trans-blot system (code n°170-3930). Thefilter was blocked with PBS-0.05 % Tween 20 overnight at roomtemperature, before incubation with the mice sera containing theanti-BASB006 antibodies. These sera are diluted 100 times in PBS-0.05 %Tween 20. and incubated on the nitrocellulose sheet for two hours atroom temperature with gentle shaking, using a mini-blotter system(Miniprotean. Bio-rad code n° 170-4017). After three repeated washingsteps in PBS-0.05% Tween 20 for 5 min., the nitrocellulose sheet isincubated at room temperature for 1 hour under gentle shaking with theappropriate conjugate (biotinylated anti-mouse Ig antibodies from sheep,Amersham code n°RPN1001) diluted at 1/500 in the same washing buffer.The membrane is washed three times as previously, and incubated for 30min. with agitation using the streptavidin-peroxidase complex (Amershamcode n°1051) diluted at 1/1000 in the washing buffer. After the lastthree repeated washing steps, the revelation occurs during the 20 minincubation time in a 50 ml solution containing 30 mg 4-chloro-1-naphtol(Sigma), 10 ml methanol, 40 ml PBS, and 30 μl of H₂O₂. The staining isstopped while washing the membrane several times in distillated water.

FIG. 4 shows recognition of the native BASB006 protein from severalNeisseria meningitidis serogroup B strains by sera from immunized mice.Results illustrated in FIG. 4 show that all strains tested present aband around 95-100 kD (see arrow), which is probably the extracellularpart of the BASB006 protein (after cleavage of the intact molecule intotwo pieces, which is known to occur in the H. influenzae Hap protein).This means that the BASB006 protein is probably expressed in most of theNeisseria meningitidis serogroup B strains. All other bands could beantibodies directed against degradation products, or againstcross-reacting antigens between E. coli and Neisseria meningitidis Bstrains, as the preparation used for immunization still contained E.coli contaminants.

Example 4 Presence of anti-BASB006 Antibodies in Sera from HumanConvalescent Patients

In this test, human convalescent sera were tested by western-blottingfor recognition of the purified recombinant BASB006 protein.

5 μg of partially purified BASB006 protein mixed with two otherNeisseria meningitidis serogroup B proteins are put into a SDS-PAGEgradient gel (4-20%, Novex, code n°EC60252) for electrophoreticmigration. Proteins are transferred to nitrocellulose sheet (0.45 μm,Bio-rad code n° 162-0114) at 100 volts for 1 hour using a Bio-radTrans-blot system (code n°170-3930). Afterwards, the filter is blockedwith PBS-0.05% Tween 20 overnight at room temperature, before incubationwith the human sera. These sera are diluted 100 times in PBS-0.05% Tween20. and incubated on the nitrocellulose sheet for two hours at roomtemperature with gentle shaking, using a mini-blotter system(Miniprotean, Bio-rad code n° 170-4017). After three repeated washingsteps in PBS-0.05 % Tween 20 for 5 min., the nitrocellulose sheet isincubated at room temperature for 11 hour under gentle shaking with theappropriate conjugate (biotinylated anti-human Ig antibodies, fromsheep, Amersham code n°RPN1003) diluted at 1/500 in the same washingbuffer. The membrane is washed three times as previously, and incubatedfor 30 min with agitation using the streptavidin-peroxidase complex(Amersham code n°1051) diluted at 1/1000 in the washing buffer. Afterthe last three repeated washing steps, the revelation occurs during the20 min incubation time in a 50 ml solution containing 30 μg4-chloro-1-naphtol (Sigma), 10 ml methanol, 40 ml of ultra-pure water,and 30μl of H₂O₂. The staining is stopped while washing the membraneseveral times in distillated water.

Results illustrated in FIG. 5 (Part B) show that all convalescent serareact against the intact BASB006 protein at around 160 kD, while 3 outof 7 convalescent sera are reacting against the possible processedBASB006 protein (+/−95-100 kD). The BASB006 bands are clearly visible atthese two molecular weights ( 95-100 and 160 kD). In part A of thewestern-blot, it can be seen that mice sera (mixture of specificantibodies against three different Ag candidates) recognize the intactrecombinant BASB006 protein at the same molecular weight, while at thelower MW, it is more difficult to discriminate which of the two bandsaround 95 kD is related to the processed BASB006 protein.

Deposited Materials

A deposit containing a Neisseria meningitidis Scrogroup B strain hasbeen deposited with the American Type Culture Collection (herein “ATCC”)on June 22, 1997 and assigned deposit number 13090. The deposit wasdescribed as Neisseria meningitidis (Albrecht and Ghon) and is afreeze-dried, 1.5-2.9 kb insert library constructed from N. meningitidisisolate. The deposit is described in Int. Bull. Bacteriol. Nomencl.Taxon. 8: 1-15 (1958).

The Neissria meningitidis strain deposit is referred to herein as “thedeposited strain” or as “the DNA of the deposited strain.”

The deposited strain contains the full length BASB006 gene. The sequenceof the polynucleotides contained in the deposited strain, as well as theamino acid sequence of any polypeptide encoded thereby, are controllingin the event of any conflict with any description of sequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. §112.

6 1 4365 DNA Bacteria 1 atgaaaacaa ccgacaaacg gacaaccgaa acacaccgcaaagccccgaa aaccggtcgc 60 atccgcttct cgcctgctta cttagccata tgcctgtcgttcggcattct tccccaagcc 120 tgggcgggac acacttattt cggcatcaac taccaatactatcgcgactt tgccgaaaat 180 aaaggcaagt ttgcagtcgg ggcgaaagat attgaggtttacaacaaaaa aggggagttg 240 gtcggcaaat caatgacaaa agccccgatg attgatttttctgtggtgtc gcgtaacggc 300 gtggcggcat tggtgggcga tcaatatatt gtgagcgtggcacataacgg cggctataac 360 aacgttgatt ttggtgcgga gggaagcaat cccgatcagcaccgtttttc ttatcaaatt 420 gtgaaaagaa ataattataa agcagggact aacggtcatccttatggtgg cgattatcat 480 atgccgcgtt tacataaatt tgtaaccgat gcagaacctgttgaaatgac cagttatatg 540 gatgggcgga aatatatcga tcaaaataat taccctgaccgtgttcgtat tggggcaggc 600 aggcaatatt ggcgatctga tgaagatgag cccaataaccgcgaaagttc atatcatatt 660 gcaagtgcgt attcttggct cgttggtggc aatacctttgcacaaaatgg atcaggtggt 720 ggcacagtca acttaggtag tgaaaaaatt aaacatagcccatatggttt tttaccaaca 780 ggaggctcat ttggcgacag tggctcacca atgtttatctatgatgccca aaagcaaaag 840 tggttaatta atggggtatt gcaaacgggc aacccctatataggaaaaag caatggcttc 900 cagctggttc gtaaagattg gttctatgat gaaatctttgctggagatac ccattcagta 960 ttctacgaac cacatcaaaa tgggaaatac acttttcacgacaataataa tggcacagga 1020 aaaatcaatg ccaaacatga acacaattct ctgcctaatagattaaaaac acgaaccgtt 1080 caattgttta atgtttcttt atccgagaca gcaagagaacctgtttatca tgctgcaggt 1140 ggtgtcaaca gttatcgacc cagactgaat aatggagaaaatatttcctt tattgacgaa 1200 ggaaaaggcg aattgatact taccagcaac atcaatcaaggtgctggagg attatatttc 1260 caaggagatt ttacggtctc gcctgaaaat aacgaaacgtggcaaggtgc gggcgttcat 1320 atcagtgaag acagtaccgt tacttggaaa gtaaacggcgtggcaaacga ccgcctgtcc 1380 aaaatcggca aaggcacgct gcacgttcaa gccaaaggggaaaaccaagg ctcgatcagc 1440 gtgggcgacg gtaaagttat tttagatcaa caagcagatgaaaataataa aaaacaagcc 1500 tttagtgaaa tcggcttggt cagcggcagg ggtacggtgcaactgaatgc cgataatcag 1560 ttcaaccccg acaaactcta tttcggcttt cgcggcggacgtttggattt gaacgggcat 1620 tcgctttcgt tccaccgtat tcaaaatacc gatgaaggggcgatgattgt caaccacaat 1680 caagacaaag aatccaccgt taccattaca ggcaataaagatattgctac aaccggcaat 1740 aacaacagct tggatagcaa aaaagaaatt gcctacaacggttggtttgg cgagaaagat 1800 acgaccaaaa cgaacgggcg gctcaacctt gtttaccagcccgccgcaga agaccgcacc 1860 ctgctgcttt ccggcggaac aaatttaaac ggtaacatcacgcaaacaaa cggcaaactg 1920 tttttcagcg gcagaccgac accgcacgcc tacaatcatttaggaagcgg gtggtcaaaa 1980 atggaaggta tcccacaagg agaaatcgtg tgggacaacgactggatcaa ccgcacgttt 2040 aaagcggaaa atttccatat tcagggcggg caggcggtgatttcccgcaa tgttgccaaa 2100 gtggaaggcg attggcattt gagcaatcac gcccaagcagtttttggtgt cgcaccgcat 2160 caaagccaca caatctgtac acgttcggac tggacgggtctgacaaattg tgtcgaaaaa 2220 accattaccg acgataaagt gattgcttca ttgactaagaccgacatcag cggcaatgtc 2280 agccttgccg atcacgctca tttaaatctc acagggcttgccacactcaa cggcaatctt 2340 agtgcaaatg gcgatacacg ttatacagtc agccacaacgccacccaaaa cggcgacctt 2400 agcctcgtgg gcaatgccca agcaacattt aatcaagccacattaaacgg caacacatcg 2460 gcttcgggca atgcttcatt taatctaagc aacaacgccgtacaaaacgg cagtctgacg 2520 ctttccggca acgctaaggc aaacgtaagc cattccgcactcaacggtaa tgtctcccta 2580 gccgataagg cagtattcca ttttgaaagc agccgctttaccggacaaat cagcggcagc 2640 aaggatacgg cattacactt aaaagacagc gaatggacgctgccgtcagg cacggaatta 2700 ggcaatttaa accttgacaa cgccaccatt acactcaattccgcctatcg ccacgatgcg 2760 gcaggggcgc aaaccggcag tgcgacagat gcgccgcgccgccgttcgcg ccgttcccta 2820 ttatccgtta cacctccggc ttcggcagaa tcccatttcaacacgctgac ggtaaacggc 2880 aaattgaacg gtcagggaac attccgcttt atgtcggaactcttcggcta ccgaagcgac 2940 aaattgaagc tggcggaaag ttccgaaggc acttacaccttggcggtcaa caataccggc 3000 aacgaacccg taagcctcga tcaattgacg gtagtggaagggaaagacaa caaaccgctg 3060 tccgaaaacc ttaatttcac cctgcaaaac gaacacgtcgatgccggcgc gtggcgttac 3120 caactcatcc gcaaagacgg cgagttccgc ctgcataatccggtcaaaga acaagagctt 3180 tccgacaaac tcggcaaggc agaagccaaa aaacaggcgggaaaagacaa cgcgcaaagc 3240 cttgacgcgc tgattgcggc cgggcgcgat gccgtcgaaaagacagaaag cgttgccgaa 3300 ccggcccggc aggcaggcgg ggaaaatgtc ggcattatgcaggcggagga agagaaaaaa 3360 cgggtgcagg cggataaaga caccgccttg gcgaaacagcgcgaagggaa aacccggccg 3420 gctaccaccg ccttcccccg cgcccgccgc gcccgccgggatttgccgca accgcagccc 3480 caaccgcaac cccaaccgca gcgcgacctg atcagccgttatgccaatag cggtttgagt 3540 gaattttccg ccacgctcaa cagcgttttc gccgtacaggacgaattaga ccgcgtattt 3600 gccgaagacc gccgcaacgc cgtttggaca agcggcatccgggacaccaa acactaccgt 3660 tcgcaagatt tccgcgccta ccgccaacaa accgacctgcgccaaatcgg tatgcagaaa 3720 aacctcggca gcgggcgcgt cggcatcctg ttttcgcacaaccggaccga aaacaccttc 3780 gacgacggca tcggcaactc ggcacggctt gcccacggcgccgttttcgg gcaatacggc 3840 atcggcaggt tcgacatcgg catcagcacg ggcgcgggttttagcagcgg cagtctttca 3900 gacgacatcg gaagcaaaat ccgccgccgc gtgctgcattacggcattca ggcacgatac 3960 cgcgccggtt tcggcggatt cggcatcgaa ccgcacatcggcgcaacgcg ctatttcgtc 4020 caaaaagcgg attaccgcta cgaaaacgtc aatatcgccacccccggcct tgcgttcaac 4080 cgctaccgcg cgggcattaa ggcagattat tcattcaaaccggcgcaaca catttccatc 4140 acgccttatt tgagcctgtc ctataccgat gccgcttcgggcaaagtccg aacgcgcgtc 4200 aataccgccg tattggctca ggatttcggc aaaacccgcagtgcggaatg gggcgtaaac 4260 gccgaaatca aaggtttcac gctgtccctc cacgctgccgccgccaaagg cccgcaactg 4320 gaagcgcaac acagcgcggg catcaaatta ggctaccgctggtaa 4365 2 1454 PRT Bacteria 2 Met Lys Thr Thr Asp Lys Arg Thr Thr GluThr His Arg Lys Ala Pro 1 5 10 15 Lys Thr Gly Arg Ile Arg Phe Ser ProAla Tyr Leu Ala Ile Cys Leu 20 25 30 Ser Phe Gly Ile Leu Pro Gln Ala TrpAla Gly His Thr Tyr Phe Gly 35 40 45 Ile Asn Tyr Gln Tyr Tyr Arg Asp PheAla Glu Asn Lys Gly Lys Phe 50 55 60 Ala Val Gly Ala Lys Asp Ile Glu ValTyr Asn Lys Lys Gly Glu Leu 65 70 75 80 Val Gly Lys Ser Met Thr Lys AlaPro Met Ile Asp Phe Ser Val Val 85 90 95 Ser Arg Asn Gly Val Ala Ala LeuVal Gly Asp Gln Tyr Ile Val Ser 100 105 110 Val Ala His Asn Gly Gly TyrAsn Asn Val Asp Phe Gly Ala Glu Gly 115 120 125 Ser Asn Pro Asp Gln HisArg Phe Ser Tyr Gln Ile Val Lys Arg Asn 130 135 140 Asn Tyr Lys Ala GlyThr Asn Gly His Pro Tyr Gly Gly Asp Tyr His 145 150 155 160 Met Pro ArgLeu His Lys Phe Val Thr Asp Ala Glu Pro Val Glu Met 165 170 175 Thr SerTyr Met Asp Gly Arg Lys Tyr Ile Asp Gln Asn Asn Tyr Pro 180 185 190 AspArg Val Arg Ile Gly Ala Gly Arg Gln Tyr Trp Arg Ser Asp Glu 195 200 205Asp Glu Pro Asn Asn Arg Glu Ser Ser Tyr His Ile Ala Ser Ala Tyr 210 215220 Ser Trp Leu Val Gly Gly Asn Thr Phe Ala Gln Asn Gly Ser Gly Gly 225230 235 240 Gly Thr Val Asn Leu Gly Ser Glu Lys Ile Lys His Ser Pro TyrGly 245 250 255 Phe Leu Pro Thr Gly Gly Ser Phe Gly Asp Ser Gly Ser ProMet Phe 260 265 270 Ile Tyr Asp Ala Gln Lys Gln Lys Trp Leu Ile Asn GlyVal Leu Gln 275 280 285 Thr Gly Asn Pro Tyr Ile Gly Lys Ser Asn Gly PheGln Leu Val Arg 290 295 300 Lys Asp Trp Phe Tyr Asp Glu Ile Phe Ala GlyAsp Thr His Ser Val 305 310 315 320 Phe Tyr Glu Pro His Gln Asn Gly LysTyr Thr Phe His Asp Asn Asn 325 330 335 Asn Gly Thr Gly Lys Ile Asn AlaLys His Glu His Asn Ser Leu Pro 340 345 350 Asn Arg Leu Lys Thr Arg ThrVal Gln Leu Phe Asn Val Ser Leu Ser 355 360 365 Glu Thr Ala Arg Glu ProVal Tyr His Ala Ala Gly Gly Val Asn Ser 370 375 380 Tyr Arg Pro Arg LeuAsn Asn Gly Glu Asn Ile Ser Phe Ile Asp Glu 385 390 395 400 Gly Lys GlyGlu Leu Ile Leu Thr Ser Asn Ile Asn Gln Gly Ala Gly 405 410 415 Gly LeuTyr Phe Gln Gly Asp Phe Thr Val Ser Pro Glu Asn Asn Glu 420 425 430 ThrTrp Gln Gly Ala Gly Val His Ile Ser Glu Asp Ser Thr Val Thr 435 440 445Trp Lys Val Asn Gly Val Ala Asn Asp Arg Leu Ser Lys Ile Gly Lys 450 455460 Gly Thr Leu His Val Gln Ala Lys Gly Glu Asn Gln Gly Ser Ile Ser 465470 475 480 Val Gly Asp Gly Lys Val Ile Leu Asp Gln Gln Ala Asp Glu AsnAsn 485 490 495 Lys Lys Gln Ala Phe Ser Glu Ile Gly Leu Val Ser Gly ArgGly Thr 500 505 510 Val Gln Leu Asn Ala Asp Asn Gln Phe Asn Pro Asp LysLeu Tyr Phe 515 520 525 Gly Phe Arg Gly Gly Arg Leu Asp Leu Asn Gly HisSer Leu Ser Phe 530 535 540 His Arg Ile Gln Asn Thr Asp Glu Gly Ala MetIle Val Asn His Asn 545 550 555 560 Gln Asp Lys Glu Ser Thr Val Thr IleThr Gly Asn Lys Asp Ile Ala 565 570 575 Thr Thr Gly Asn Asn Asn Ser LeuAsp Ser Lys Lys Glu Ile Ala Tyr 580 585 590 Asn Gly Trp Phe Gly Glu LysAsp Thr Thr Lys Thr Asn Gly Arg Leu 595 600 605 Asn Leu Val Tyr Gln ProAla Ala Glu Asp Arg Thr Leu Leu Leu Ser 610 615 620 Gly Gly Thr Asn LeuAsn Gly Asn Ile Thr Gln Thr Asn Gly Lys Leu 625 630 635 640 Phe Phe SerGly Arg Pro Thr Pro His Ala Tyr Asn His Leu Gly Ser 645 650 655 Gly TrpSer Lys Met Glu Gly Ile Pro Gln Gly Glu Ile Val Trp Asp 660 665 670 AsnAsp Trp Ile Asn Arg Thr Phe Lys Ala Glu Asn Phe His Ile Gln 675 680 685Gly Gly Gln Ala Val Ile Ser Arg Asn Val Ala Lys Val Glu Gly Asp 690 695700 Trp His Leu Ser Asn His Ala Gln Ala Val Phe Gly Val Ala Pro His 705710 715 720 Gln Ser His Thr Ile Cys Thr Arg Ser Asp Trp Thr Gly Leu ThrAsn 725 730 735 Cys Val Glu Lys Thr Ile Thr Asp Asp Lys Val Ile Ala SerLeu Thr 740 745 750 Lys Thr Asp Ile Ser Gly Asn Val Ser Leu Ala Asp HisAla His Leu 755 760 765 Asn Leu Thr Gly Leu Ala Thr Leu Asn Gly Asn LeuSer Ala Asn Gly 770 775 780 Asp Thr Arg Tyr Thr Val Ser His Asn Ala ThrGln Asn Gly Asp Leu 785 790 795 800 Ser Leu Val Gly Asn Ala Gln Ala ThrPhe Asn Gln Ala Thr Leu Asn 805 810 815 Gly Asn Thr Ser Ala Ser Gly AsnAla Ser Phe Asn Leu Ser Asn Asn 820 825 830 Ala Val Gln Asn Gly Ser LeuThr Leu Ser Gly Asn Ala Lys Ala Asn 835 840 845 Val Ser His Ser Ala LeuAsn Gly Asn Val Ser Leu Ala Asp Lys Ala 850 855 860 Val Phe His Phe GluSer Ser Arg Phe Thr Gly Gln Ile Ser Gly Ser 865 870 875 880 Lys Asp ThrAla Leu His Leu Lys Asp Ser Glu Trp Thr Leu Pro Ser 885 890 895 Gly ThrGlu Leu Gly Asn Leu Asn Leu Asp Asn Ala Thr Ile Thr Leu 900 905 910 AsnSer Ala Tyr Arg His Asp Ala Ala Gly Ala Gln Thr Gly Ser Ala 915 920 925Thr Asp Ala Pro Arg Arg Arg Ser Arg Arg Ser Leu Leu Ser Val Thr 930 935940 Pro Pro Ala Ser Ala Glu Ser His Phe Asn Thr Leu Thr Val Asn Gly 945950 955 960 Lys Leu Asn Gly Gln Gly Thr Phe Arg Phe Met Ser Glu Leu PheGly 965 970 975 Tyr Arg Ser Asp Lys Leu Lys Leu Ala Glu Ser Ser Glu GlyThr Tyr 980 985 990 Thr Leu Ala Val Asn Asn Thr Gly Asn Glu Pro Val SerLeu Asp Gln 995 1000 1005 Leu Thr Val Val Glu Gly Lys Asp Asn Lys ProLeu Ser Glu Asn Leu 1010 1015 1020 Asn Phe Thr Leu Gln Asn Glu His ValAsp Ala Gly Ala Trp Arg Tyr 1025 1030 1035 1040 Gln Leu Ile Arg Lys AspGly Glu Phe Arg Leu His Asn Pro Val Lys 1045 1050 1055 Glu Gln Glu LeuSer Asp Lys Leu Gly Lys Ala Glu Ala Lys Lys Gln 1060 1065 1070 Ala GlyLys Asp Asn Ala Gln Ser Leu Asp Ala Leu Ile Ala Ala Gly 1075 1080 1085Arg Asp Ala Val Glu Lys Thr Glu Ser Val Ala Glu Pro Ala Arg Gln 10901095 1100 Ala Gly Gly Glu Asn Val Gly Ile Met Gln Ala Glu Glu Glu LysLys 1105 1110 1115 1120 Arg Val Gln Ala Asp Lys Asp Thr Ala Leu Ala LysGln Arg Glu Gly 1125 1130 1135 Lys Thr Arg Pro Ala Thr Thr Ala Phe ProArg Ala Arg Arg Ala Arg 1140 1145 1150 Arg Asp Leu Pro Gln Pro Gln ProGln Pro Gln Pro Gln Pro Gln Arg 1155 1160 1165 Asp Leu Ile Ser Arg TyrAla Asn Ser Gly Leu Ser Glu Phe Ser Ala 1170 1175 1180 Thr Leu Asn SerVal Phe Ala Val Gln Asp Glu Leu Asp Arg Val Phe 1185 1190 1195 1200 AlaGlu Asp Arg Arg Asn Ala Val Trp Thr Ser Gly Ile Arg Asp Thr 1205 12101215 Lys His Tyr Arg Ser Gln Asp Phe Arg Ala Tyr Arg Gln Gln Thr Asp1220 1225 1230 Leu Arg Gln Ile Gly Met Gln Lys Asn Leu Gly Ser Gly ArgVal Gly 1235 1240 1245 Ile Leu Phe Ser His Asn Arg Thr Glu Asn Thr PheAsp Asp Gly Ile 1250 1255 1260 Gly Asn Ser Ala Arg Leu Ala His Gly AlaVal Phe Gly Gln Tyr Gly 1265 1270 1275 1280 Ile Gly Arg Phe Asp Ile GlyIle Ser Thr Gly Ala Gly Phe Ser Ser 1285 1290 1295 Gly Ser Leu Ser AspAsp Ile Gly Ser Lys Ile Arg Arg Arg Val Leu 1300 1305 1310 His Tyr GlyIle Gln Ala Arg Tyr Arg Ala Gly Phe Gly Gly Phe Gly 1315 1320 1325 IleGlu Pro His Ile Gly Ala Thr Arg Tyr Phe Val Gln Lys Ala Asp 1330 13351340 Tyr Arg Tyr Glu Asn Val Asn Ile Ala Thr Pro Gly Leu Ala Phe Asn1345 1350 1355 1360 Arg Tyr Arg Ala Gly Ile Lys Ala Asp Tyr Ser Phe LysPro Ala Gln 1365 1370 1375 His Ile Ser Ile Thr Pro Tyr Leu Ser Leu SerTyr Thr Asp Ala Ala 1380 1385 1390 Ser Gly Lys Val Arg Thr Arg Val AsnThr Ala Val Leu Ala Gln Asp 1395 1400 1405 Phe Gly Lys Thr Arg Ser AlaGlu Trp Gly Val Asn Ala Glu Ile Lys 1410 1415 1420 Gly Phe Thr Leu SerLeu His Ala Ala Ala Ala Lys Gly Pro Gln Leu 1425 1430 1435 1440 Glu AlaGln His Ser Ala Gly Ile Lys Leu Gly Tyr Arg Trp 1445 1450 3 4374 DNABacteria 3 atgaaaacaa ccgacaaacg gacaaccgaa acacaccgca aagccccgaaaaccggccgc 60 atccgcttct cgcctgctta cttagccata tgcctgtcgt tcggcattcttccccaagcc 120 tgggcgggac acacttattt cggcatcaac taccaatact atcgcgactttgccgaaaat 180 aaaggcaagt ttgcagtcgg ggcgaaagat attgaggttt acaacaaaaaaggggagttg 240 gtcggcaaat caatgacaaa agccccgatg attgattttt ctgtggtgtcgcgtaacggc 300 gtggcggcat tggtgggcga tcaatatatt gtgagcgtgg cacataacggcggctataac 360 aacgttgatt ttggtgcgga aggaagaaat cccgatcaac atcgttttacttataaaatt 420 gtgaaacgga ataattataa agcagggact aaaggccatc cttatggtggcgattatcat 480 atgccgcgtt tacataaatt tgtcacagat gcagaacctg ttgaaatgaccagttatatg 540 gatgggcgga aatatatcga tcaaaataat taccctgacc gtgttcgtattggggcaggc 600 aggcaatatt ggcgatctga tgaagatgag cccaataacc gcgaaagttcatatcatatt 660 gcaagtgcgt attcttggct cgttggtggc aatacctttg cacaaaatggatcaggtggt 720 ggcacagtca acttaggtag tgaaaaaatt aaacatagcc catatggttttttaccaaca 780 ggaggctcat ttggcgacag tggctcacca atgtttatct atgatgcccaaaagcaaaag 840 tggttaatta atggggtatt gcaaacgggc aacccctata taggaaaaagcaatggcttc 900 cagctggttc gtaaagattg gttctatgat gaaatctttg ctggagatacccattcagta 960 ttctacgaac cacgtcaaaa tgggaaatac tcttttaacg acgataataatggcacagga 1020 aaaatcaatg ccaaacatga acacaattct ctgcctaata gattaaaaacacgaaccgtt 1080 caattgttta atgtttcttt atccgagaca gcaagagaac ctgtttatcatgctgcaggt 1140 ggtgtcaaca gttatcgacc cagactgaat aatggagaaa atatttcctttattgacgaa 1200 ggaaaaggcg aattgatact taccagcaac atcaatcaag gtgctggaggattatatttc 1260 caaggagatt ttacggtctc gcctgaaaat aacgaaacgt ggcaaggtgcgggcgttcat 1320 atcagtgaag acagtaccgt tacttggaaa gtaaacggcg tggcaaacgaccgcctgtcc 1380 aaaatcggca aaggcacgct gcacgttcaa gccaaagggg aaaaccaaggctcgatcagc 1440 gtgggcgacg gtacagtcat tttggatcag caggcagacg ataaaggcaaaaaacaagcc 1500 tttagtgaaa tcggcttggt cagcggcagg ggtacggtgc aactgaatgccgataatcag 1560 ttcaaccccg acaaactcta tttcggcttt cgcggcggac gtttggatttaaacgggcat 1620 tcgctttcgt tccaccgtat tcaaaatacc gatgaagggg cgatgattgtcaaccacaat 1680 caagacaaag aatccaccgt taccattaca ggcaataaag atattgctacaaccggcaat 1740 aacaacagct tggatagcaa aaaagaaatt gcctacaacg gttggtttggcgagaaagat 1800 acgaccaaaa cgaacgggcg gctcaacctt gtttaccagc ccgccgcagaagaccgcacc 1860 ctgctgcttt ccggcggaac aaatttaaac ggcaacatca cgcaaacaaacggcaaactg 1920 tttttcagcg gcagaccaac accgcacgcc tacaatcatt taaacgaccattggtcgcaa 1980 aaagagggca ttccacgcgg ggaaatcgtg tgggacaacg actggatcaaccgcacattt 2040 aaagcggaaa acttccaaat taaaggcgga caggcggtgg tttcccgcaatgttgccaaa 2100 gtgaaaggcg attggcattt gagcaatcac gcccaagcag tttttggtgtcgcaccgcat 2160 caaagccaca caatctgtac acgttcggac tggacgggtc tgacaaattgtgtcgaaaaa 2220 accattaccg acgataaagt gattgcttca ttgactaaga ccgacatcagcggcaatgtc 2280 gatcttgccg atcacgctca tttaaatctc acagggcttg ccacactcaacggcaatctt 2340 agtgcaaatg gcgatacacg ttatacagtc agccacaacg ccacccaaaacggcaacctt 2400 agcctcgtgg gcaatgccca agcaacattt aatcaagcca cattaaacggcaacacatcg 2460 gcttcgggca atgcttcatt taatctaagc gaccacgccg tacaaaacggcagtctgacg 2520 ctttccggca acgctaaggc aaacgtaagc cattccgcac tcaacggtaatgtctcccta 2580 gccgataagg cagtattcca ttttgaaagc agccgcttta ccggacaaatcagcggcggc 2640 aaggatacgg cattacactt aaaagacagc gaatggacgc tgccgtcaggcacggaatta 2700 ggcaatttaa accttgacaa cgccaccatt acactcaatt ccgcctatcgccacgatgcg 2760 gcaggggcgc aaaccggcag tgcgacagat gcgccgcgcc gccgttcgcgccgttcgcgc 2820 cgttccctat tatccgttac accgccaact tcggtagaat cccgtttcaacacgctgacg 2880 gtaaacggca aattgaacgg tcagggaaca ttccgcttta tgtcggaactcttcggctac 2940 cgcagcgaca aattgaagct ggcggaaagt tccgaaggca cttacaccttggcggtcaac 3000 aataccggca acgaacctgc aagccttgaa caattgacgg tagtggaaggaaaagacaac 3060 aaaccgctgt ccgaaaactt taatttcacc ttgcaaaacg aacacgtcgatgccggcgcg 3120 tggcgttacc aactcatccg caaagacggc gagttccgcc tgcataatccggtcaaagaa 3180 caagagcttt ccgacaaact cggcaaggca gaagccaaaa aacaggcggaaaaagacaac 3240 gcgcaaagcc ttgacgcgct gattgcggcc gggcgcgatg ccgtcgaaaagacagaaagc 3300 gttgccgaac cggcccggca ggcaggcggg gaaaatgtcg gcattatgcaggcggaggaa 3360 gagaaaaaac gggtgcaggc ggataaagac accgccttgg cgaaacagcgcgaagcggaa 3420 acccggccgg ctaccaccgc cttcccccgc gcccgccgcg cccgccgggatttgccgcaa 3480 ctgcaacccc aaccgcagcc ccaaccgcag cgcgacctga tcagccgttatgccaatagc 3540 ggtttgagtg aattttccgc cacgctcaac agcgttttcg ccgtacaggacgaattagac 3600 cgcgtatttg ccgaagaacg ccgcaacgcc gtttggacaa gcggcatccgggacaccaaa 3660 cactaccgtt cgcaagattt ccgcgcctac cgccaacaaa ccgacctgcgccaaatcggt 3720 atgcagaaaa acctcggcag cgggcgcgtc ggcatcctgt tttcgcacaaccggaccgaa 3780 aacaccttcg acgacggcat cggcaactcg gcacggcttg cccacggcgccgttttcggg 3840 caatacggca tcgacaggtt ctacatcggc atcagcgcgg gcgcgggttttagcagcggc 3900 agcctttcag acggcatcgg aggcaaaatc cgccgccgcg tgctgcattacggcattcag 3960 gcacgatacc gcgccggttt cggcggattc ggcatcgaac cgcacatcggcgcaacgcgc 4020 tatttcgtcc aaaaagcgga ttaccgctac gaaaacgtca atatcgccacccccggcctt 4080 gcattcaacc gctaccgcgc gggcattaag gcagattatt cattcaaaccggcgcaacac 4140 atttccatca cgccttattt gagcctgtcc tataccgatg ccgcttcgggcaaagtccga 4200 acacgcgtca ataccgccgt attggctcag gatttcggca aaacccgcagtgcggaatgg 4260 ggcgtaaacg ccgaaatcaa aggcttcacg ctgtccctcc acgctgccgccgccaaaggc 4320 ccgcaactgg aagcgcaaca cagcgcgggc atcaaattag gctaccgctggtaa 4374 4 1457 PRT Bacteria 4 Met Lys Thr Thr Asp Lys Arg Thr Thr GluThr His Arg Lys Ala Pro 1 5 10 15 Lys Thr Gly Arg Ile Arg Phe Ser ProAla Tyr Leu Ala Ile Cys Leu 20 25 30 Ser Phe Gly Ile Leu Pro Gln Ala TrpAla Gly His Thr Tyr Phe Gly 35 40 45 Ile Asn Tyr Gln Tyr Tyr Arg Asp PheAla Glu Asn Lys Gly Lys Phe 50 55 60 Ala Val Gly Ala Lys Asp Ile Glu ValTyr Asn Lys Lys Gly Glu Leu 65 70 75 80 Val Gly Lys Ser Met Thr Lys AlaPro Met Ile Asp Phe Ser Val Val 85 90 95 Ser Arg Asn Gly Val Ala Ala LeuVal Gly Asp Gln Tyr Ile Val Ser 100 105 110 Val Ala His Asn Gly Gly TyrAsn Asn Val Asp Phe Gly Ala Glu Gly 115 120 125 Arg Asn Pro Asp Gln HisArg Phe Thr Tyr Lys Ile Val Lys Arg Asn 130 135 140 Asn Tyr Lys Ala GlyThr Lys Gly His Pro Tyr Gly Gly Asp Tyr His 145 150 155 160 Met Pro ArgLeu His Lys Phe Val Thr Asp Ala Glu Pro Val Glu Met 165 170 175 Thr SerTyr Met Asp Gly Arg Lys Tyr Ile Asp Gln Asn Asn Tyr Pro 180 185 190 AspArg Val Arg Ile Gly Ala Gly Arg Gln Tyr Trp Arg Ser Asp Glu 195 200 205Asp Glu Pro Asn Asn Arg Glu Ser Ser Tyr His Ile Ala Ser Ala Tyr 210 215220 Ser Trp Leu Val Gly Gly Asn Thr Phe Ala Gln Asn Gly Ser Gly Gly 225230 235 240 Gly Thr Val Asn Leu Gly Ser Glu Lys Ile Lys His Ser Pro TyrGly 245 250 255 Phe Leu Pro Thr Gly Gly Ser Phe Gly Asp Ser Gly Ser ProMet Phe 260 265 270 Ile Tyr Asp Ala Gln Lys Gln Lys Trp Leu Ile Asn GlyVal Leu Gln 275 280 285 Thr Gly Asn Pro Tyr Ile Gly Lys Ser Asn Gly PheGln Leu Val Arg 290 295 300 Lys Asp Trp Phe Tyr Asp Glu Ile Phe Ala GlyAsp Thr His Ser Val 305 310 315 320 Phe Tyr Glu Pro Arg Gln Asn Gly LysTyr Ser Phe Asn Asp Asp Asn 325 330 335 Asn Gly Thr Gly Lys Ile Asn AlaLys His Glu His Asn Ser Leu Pro 340 345 350 Asn Arg Leu Lys Thr Arg ThrVal Gln Leu Phe Asn Val Ser Leu Ser 355 360 365 Glu Thr Ala Arg Glu ProVal Tyr His Ala Ala Gly Gly Val Asn Ser 370 375 380 Tyr Arg Pro Arg LeuAsn Asn Gly Glu Asn Ile Ser Phe Ile Asp Glu 385 390 395 400 Gly Lys GlyGlu Leu Ile Leu Thr Ser Asn Ile Asn Gln Gly Ala Gly 405 410 415 Gly LeuTyr Phe Gln Gly Asp Phe Thr Val Ser Pro Glu Asn Asn Glu 420 425 430 ThrTrp Gln Gly Ala Gly Val His Ile Ser Glu Asp Ser Thr Val Thr 435 440 445Trp Lys Val Asn Gly Val Ala Asn Asp Arg Leu Ser Lys Ile Gly Lys 450 455460 Gly Thr Leu His Val Gln Ala Lys Gly Glu Asn Gln Gly Ser Ile Ser 465470 475 480 Val Gly Asp Gly Thr Val Ile Leu Asp Gln Gln Ala Asp Asp LysGly 485 490 495 Lys Lys Gln Ala Phe Ser Glu Ile Gly Leu Val Ser Gly ArgGly Thr 500 505 510 Val Gln Leu Asn Ala Asp Asn Gln Phe Asn Pro Asp LysLeu Tyr Phe 515 520 525 Gly Phe Arg Gly Gly Arg Leu Asp Leu Asn Gly HisSer Leu Ser Phe 530 535 540 His Arg Ile Gln Asn Thr Asp Glu Gly Ala MetIle Val Asn His Asn 545 550 555 560 Gln Asp Lys Glu Ser Thr Val Thr IleThr Gly Asn Lys Asp Ile Ala 565 570 575 Thr Thr Gly Asn Asn Asn Ser LeuAsp Ser Lys Lys Glu Ile Ala Tyr 580 585 590 Asn Gly Trp Phe Gly Glu LysAsp Thr Thr Lys Thr Asn Gly Arg Leu 595 600 605 Asn Leu Val Tyr Gln ProAla Ala Glu Asp Arg Thr Leu Leu Leu Ser 610 615 620 Gly Gly Thr Asn LeuAsn Gly Asn Ile Thr Gln Thr Asn Gly Lys Leu 625 630 635 640 Phe Phe SerGly Arg Pro Thr Pro His Ala Tyr Asn His Leu Asn Asp 645 650 655 His TrpSer Gln Lys Glu Gly Ile Pro Arg Gly Glu Ile Val Trp Asp 660 665 670 AsnAsp Trp Ile Asn Arg Thr Phe Lys Ala Glu Asn Phe Gln Ile Lys 675 680 685Gly Gly Gln Ala Val Val Ser Arg Asn Val Ala Lys Val Lys Gly Asp 690 695700 Trp His Leu Ser Asn His Ala Gln Ala Val Phe Gly Val Ala Pro His 705710 715 720 Gln Ser His Thr Ile Cys Thr Arg Ser Asp Trp Thr Gly Leu ThrAsn 725 730 735 Cys Val Glu Lys Thr Ile Thr Asp Asp Lys Val Ile Ala SerLeu Thr 740 745 750 Lys Thr Asp Ile Ser Gly Asn Val Asp Leu Ala Asp HisAla His Leu 755 760 765 Asn Leu Thr Gly Leu Ala Thr Leu Asn Gly Asn LeuSer Ala Asn Gly 770 775 780 Asp Thr Arg Tyr Thr Val Ser His Asn Ala ThrGln Asn Gly Asn Leu 785 790 795 800 Ser Leu Val Gly Asn Ala Gln Ala ThrPhe Asn Gln Ala Thr Leu Asn 805 810 815 Gly Asn Thr Ser Ala Ser Gly AsnAla Ser Phe Asn Leu Ser Asp His 820 825 830 Ala Val Gln Asn Gly Ser LeuThr Leu Ser Gly Asn Ala Lys Ala Asn 835 840 845 Val Ser His Ser Ala LeuAsn Gly Asn Val Ser Leu Ala Asp Lys Ala 850 855 860 Val Phe His Phe GluSer Ser Arg Phe Thr Gly Gln Ile Ser Gly Gly 865 870 875 880 Lys Asp ThrAla Leu His Leu Lys Asp Ser Glu Trp Thr Leu Pro Ser 885 890 895 Gly ThrGlu Leu Gly Asn Leu Asn Leu Asp Asn Ala Thr Ile Thr Leu 900 905 910 AsnSer Ala Tyr Arg His Asp Ala Ala Gly Ala Gln Thr Gly Ser Ala 915 920 925Thr Asp Ala Pro Arg Arg Arg Ser Arg Arg Ser Arg Arg Ser Leu Leu 930 935940 Ser Val Thr Pro Pro Thr Ser Val Glu Ser Arg Phe Asn Thr Leu Thr 945950 955 960 Val Asn Gly Lys Leu Asn Gly Gln Gly Thr Phe Arg Phe Met SerGlu 965 970 975 Leu Phe Gly Tyr Arg Ser Asp Lys Leu Lys Leu Ala Glu SerSer Glu 980 985 990 Gly Thr Tyr Thr Leu Ala Val Asn Asn Thr Gly Asn GluPro Ala Ser 995 1000 1005 Leu Glu Gln Leu Thr Val Val Glu Gly Lys AspAsn Lys Pro Leu Ser 1010 1015 1020 Glu Asn Phe Asn Phe Thr Leu Gln AsnGlu His Val Asp Ala Gly Ala 1025 1030 1035 1040 Trp Arg Tyr Gln Leu IleArg Lys Asp Gly Glu Phe Arg Leu His Asn 1045 1050 1055 Pro Val Lys GluGln Glu Leu Ser Asp Lys Leu Gly Lys Ala Glu Ala 1060 1065 1070 Lys LysGln Ala Glu Lys Asp Asn Ala Gln Ser Leu Asp Ala Leu Ile 1075 1080 1085Ala Ala Gly Arg Asp Ala Val Glu Lys Thr Glu Ser Val Ala Glu Pro 10901095 1100 Ala Arg Gln Ala Gly Gly Glu Asn Val Gly Ile Met Gln Ala GluGlu 1105 1110 1115 1120 Glu Lys Lys Arg Val Gln Ala Asp Lys Asp Thr AlaLeu Ala Lys Gln 1125 1130 1135 Arg Glu Ala Glu Thr Arg Pro Ala Thr ThrAla Phe Pro Arg Ala Arg 1140 1145 1150 Arg Ala Arg Arg Asp Leu Pro GlnLeu Gln Pro Gln Pro Gln Pro Gln 1155 1160 1165 Pro Gln Arg Asp Leu IleSer Arg Tyr Ala Asn Ser Gly Leu Ser Glu 1170 1175 1180 Phe Ser Ala ThrLeu Asn Ser Val Phe Ala Val Gln Asp Glu Leu Asp 1185 1190 1195 1200 ArgVal Phe Ala Glu Glu Arg Arg Asn Ala Val Trp Thr Ser Gly Ile 1205 12101215 Arg Asp Thr Lys His Tyr Arg Ser Gln Asp Phe Arg Ala Tyr Arg Gln1220 1225 1230 Gln Thr Asp Leu Arg Gln Ile Gly Met Gln Lys Asn Leu GlySer Gly 1235 1240 1245 Arg Val Gly Ile Leu Phe Ser His Asn Arg Thr GluAsn Thr Phe Asp 1250 1255 1260 Asp Gly Ile Gly Asn Ser Ala Arg Leu AlaHis Gly Ala Val Phe Gly 1265 1270 1275 1280 Gln Tyr Gly Ile Asp Arg PheTyr Ile Gly Ile Ser Ala Gly Ala Gly 1285 1290 1295 Phe Ser Ser Gly SerLeu Ser Asp Gly Ile Gly Gly Lys Ile Arg Arg 1300 1305 1310 Arg Val LeuHis Tyr Gly Ile Gln Ala Arg Tyr Arg Ala Gly Phe Gly 1315 1320 1325 GlyPhe Gly Ile Glu Pro His Ile Gly Ala Thr Arg Tyr Phe Val Gln 1330 13351340 Lys Ala Asp Tyr Arg Tyr Glu Asn Val Asn Ile Ala Thr Pro Gly Leu1345 1350 1355 1360 Ala Phe Asn Arg Tyr Arg Ala Gly Ile Lys Ala Asp TyrSer Phe Lys 1365 1370 1375 Pro Ala Gln His Ile Ser Ile Thr Pro Tyr LeuSer Leu Ser Tyr Thr 1380 1385 1390 Asp Ala Ala Ser Gly Lys Val Arg ThrArg Val Asn Thr Ala Val Leu 1395 1400 1405 Ala Gln Asp Phe Gly Lys ThrArg Ser Ala Glu Trp Gly Val Asn Ala 1410 1415 1420 Glu Ile Lys Gly PheThr Leu Ser Leu His Ala Ala Ala Ala Lys Gly 1425 1430 1435 1440 Pro GlnLeu Glu Ala Gln His Ser Ala Gly Ile Lys Leu Gly Tyr Arg 1445 1450 1455Trp 5 34 DNA Artificial Sequence Primer 5 gggggctagc aaaacaaccgacaaacggac aacc 34 6 40 DNA Artificial Sequence Primer 6 ggggaagcttccagcggtag cggtagccta atttgatgcc 40

What is claimed is:
 1. An isolated polypeptide comprising SEQ ID NO:2.2. The isolated polypeptide of claim 1, wherein the isolated polypeptideconsists of SEQ ID NO:2.
 3. A fusion protein comprising the isolatedpolypeptide of claim 1 and a polypeptide selected to: (a) provide Thelper epitopes or (b) facilitate purification from a recombinantexpression system.
 4. An immunogenic composition comprising the isolatedpolypeptide of claim 1 and a pharmaceutically acceptable carrier.
 5. Theimmunogenic composition of claim 4, wherein the composition comprises atleast one other Neisseria meningitidis antigen.
 6. An isolatedpolypeptide comprising SEQ ID NO:4.
 7. The isolated polypeptide of claim6, wherein the isolated polypeptide consists of SEQ ID NO:4.
 8. A fusionprotein comprising the isolated polypeptide of claim 6 and a polypeptideselected to: (a) provide T helper epitopes or (b) facilitatepurification from a recombinant expression system.
 9. An immunogeniccomposition comprising the isolated polypeptide of claim 6 and apharmaceutically acceptable carrier.
 10. The immunogenic composition ofclaim 9, wherein the composition comprises at least one other Neisseriameningitidis antigen.